Purification, reactivity with IgE and cDNA cloning of parvalbumin as the major allergen of mackerels.

Science.gov (United States)

Hamada, Y; Tanaka, H; Ishizaki, S; Ishida, M; Nagashima, Y; Shiomi, K

2003-08-01

Three species of mackerels (Scomber japonicus, S. australasicus and S. scombrus) are widely consumed and considered to be most frequently involved in incidents of IgE-mediated fish allergy in Japan. In this study, parvalbumin, a possible candidate for the major allergen, was purified from the white muscle of three species of mackerels by gel filtration on Sephadex G-75 and reverse-phase HPLC on TSKgel ODS-120T. All the purified preparations from three species gave a single band of about 11 kDa and were clearly identified as parvalbumins by analyses of their partial amino acid sequences. In ELISA experiments, four of five sera from fish-allergic patients reacted to all the purified parvalbumins, demonstrating that parvalbumin is the major allergen in common with the mackerels. Antigenic cross-reactivity among the mackerel parvalbumins was also established by ELISA inhibition experiments. A cDNA library was constructed from the white muscle of S. japonicus and the cDNA encoding parvalbumin was cloned. The amino acid sequence translated from the nucleotide sequence revealed that the S. japonicus parvalbumin is composed of 108 residues, being a member of beta-type parvalbumins.

Ectopic expression of the calcium-binding protein parvalbumin in mouse liver endothelial cells

DEFF Research Database (Denmark)

Castillo, M B; Berchtold, M W; Rülicke, T

1997-01-01

To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical...... methods have been used to detect the presence of ectopic parvalbumin expression in different tissues. Here we show the expression of parvalbumin in endothelial cells lining the liver sinusoids in situ and after isolation in vitro. The hemodynamic effects of endothelin 1, a peptide hormone mediating potent...... vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased...

The Effects of Dopamine and Estrogen upon Cortical Parvalbumin Expression

Science.gov (United States)

2001-10-01

male rat. Brain Res 646:157-160. Solodkin A, Veldhuizen SD, Van Hossen GW (1996) Contingent vulnerability of entorhinal parvalbumin-containing...in the male rat. Brain Res 646:157-160. Solodkin A, Veldhuizen SD, Van Hossen GW (1996) Contingent vulnerability of entorhinal parvalbumin-containing...neurons (Wang et al, 1995, 1996). Both pyramidal (Lewis et al, 1995) and GABAergic neurons ( van Kammen et al, 1998; Ohnuma et al, 1999; Volk et al, 2000

Expression and evaluation of IgE-binding capacity of recombinant Pacific mackerel parvalbumin

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Yuki Hamada

2004-01-01

Conclusions: Because the recombinant Pacific mackerel parvalbumin bearing the IgE-binding capacity of the natural counterpart is cross-reactive with various fish parvalbumins, it can be a useful tool for the diagnosis and immunotherapy of fish allergy.

Parvalbumin--the major tropical fish allergen.

Science.gov (United States)

Lim, Dawn Li-Chern; Neo, Keng Hwee; Yi, Fong Cheng; Chua, Kaw Yan; Goh, Denise Li-Meng; Shek, Lynette Pei-Chi; Giam, Yoke Chin; Van Bever, Hugo P S; Lee, Bee Wah

2008-08-01

Fish allergy is common in countries where consumption is high. Asian nations are amongst the world's largest consumers of fish but the allergen profiles of tropical fish are unknown. This study sought to evaluate the allergenicity of four commonly consumed tropical fish, the threadfin (Polynemus indicus), Indian anchovy (Stolephorus indicus), pomfret (Pampus chinensis) and tengirri (Scomberomorus guttatus). Immunoglobulin E (IgE) cross-reactivity with parvalbumin of cod fish (Gad c 1), the major fish allergen, was also studied. Detection of tropical fish and cod specific-IgE was performed by UniCap assay, and skin prick tests were also carried out. The IgE-binding components of tropical fish were identified using IgE immunoblot techniques, and cross-reactivity with Gad c 1 was assessed by ELISA inhibition and IgE immunoblot inhibition. Clinically, nine of 10 patients studied were allergic to multiple fish. All patients exhibited detectable specific-IgE to cod fish (10 of 10 skin prick test positive, eight of 10 UniCap assay positive) despite lack of previous exposure. The major allergen of the four tropical fish was the 12-kDa parvalbumin. IgE cross-reactivity of these allergens to Gad c 1 was observed to be moderate to high in the tropical fish studied. Parvalbumins are the major allergens in commonly consumed tropical fish. They are cross-reactive with each other as well as with Gad c 1. Commercial tests for cod fish appear to be sufficient for the detection of tropical fish specific-IgE.

A recombinant hypoallergenic parvalbumin mutant for immunotherapy of IgE-mediated fish allergy.

Science.gov (United States)

Swoboda, Ines; Bugajska-Schretter, Agnes; Linhart, Birgit; Verdino, Petra; Keller, Walter; Schulmeister, Ulrike; Sperr, Wolfgang R; Valent, Peter; Peltre, Gabriel; Quirce, Santiago; Douladiris, Nikolaos; Papadopoulos, Nikolaos G; Valenta, Rudolf; Spitzauer, Susanne

2007-05-15

IgE-mediated allergy to fish is a frequent cause of severe anaphylactic reactions. Parvalbumin, a small calcium-binding protein, is the major fish allergen. We have recently isolated a cDNA coding for carp parvalbumin, Cyp c 1, and expressed in Escherichia coli a recombinant Cyp c 1 molecule, which contained most IgE epitopes of saltwater and freshwater fish. In this study, we introduced mutations into the calcium-binding domains of carp parvalbumin by site-directed mutagenesis and produced in E. coli three parvalbumin mutants containing amino acid exchanges either in one (single mutants; Mut-CD and Mut-EF) or in both of the calcium-binding sites (double mutant; Mut-CD/EF). Circular dichroism analyses of the purified derivatives and the wild-type allergen showed that Mut-CD/EF exhibited the greatest reduction of overall protein fold. Dot blot assays and immunoblot inhibition experiments performed with sera from 21 fish-allergic patients showed that Mut-CD/EF had a 95% reduced IgE reactivity and represented the derivative with the least allergenic activity. The latter was confirmed by in vitro basophil histamine release assays and in vivo skin prick testing. The potential applicability for immunotherapy of Mut-CD/EF was demonstrated by the fact that mouse IgG Abs could be raised by immunization with the mutated molecule, which cross-reacted with parvalbumins from various fish species and inhibited the binding of fish-allergic patients' IgE to the wild-type allergen. Using the hypoallergenic carp parvalbumin mutant Mut-CD/EF, it may be possible to treat fish allergy by immunotherapy.

Fish allergens at a glance: variable allergenicity of parvalbumins, the major fish allergens.

Science.gov (United States)

Kuehn, Annette; Swoboda, Ines; Arumugam, Karthik; Hilger, Christiane; Hentges, François

2014-01-01

Fish is a common trigger of severe, food-allergic reactions. Only a limited number of proteins induce specific IgE-mediated immune reactions. The major fish allergens are the parvalbumins. They are members of the calcium-binding EF-hand protein family characterized by a conserved protein structure. They represent highly cross-reactive allergens for patients with specific IgE to conserved epitopes. These patients might experience clinical reactions with various fish species. On the other hand, some individuals have IgE antibodies directed against unique, species-specific parvalbumin epitopes, and these patients show clinical symptoms only with certain fish species. Furthermore, different parvalbumin isoforms and isoallergens are present in the same fish and might display variable allergenicity. This was shown for salmon homologs, where only a single parvalbumin (beta-1) isoform was identified as allergen in specific patients. In addition to the parvalbumins, several other fish proteins, enolases, aldolases, and fish gelatin, seem to be important allergens. New clinical and molecular insights advanced the knowledge and understanding of fish allergy in the last years. These findings were useful for the advancement of the IgE-based diagnosis and also for the management of fish allergies consisting of advice and treatment of fish-allergic patients.

Fish allergens at a glance: Variable allergenicity of parvalbumins, the major fish allergens

Directory of Open Access Journals (Sweden)

Annette eKuehn

2014-04-01

Full Text Available Fish is a common trigger of severe, food-allergic reactions. Only a limited number of proteins induce specific IgE-mediated immune reactions. The major fish allergens are the parvalbumins. They are members of the calcium-binding EF-hand protein family characterized by a conserved protein structure. They represent highly cross-reactive allergens for patients with specific IgE to conserved epitopes. These patients might experience clinical reactions with various fish species. On the other hand, some individuals have IgE antibodies directed against unique, species-specific parvalbumin epitopes, and these patients show clinical symptoms only with certain fish species. Furthermore, different parvalbumin isoforms and isoallergens are present in the same fish and might display variable allergenicity. This was shown for salmon homologs, where only a single parvalbumin (beta-1 isoform was identified as allergen in specific patients. In addition to the parvalbumins, several other fish proteins, enolases, aldolases and fish gelatin, seem to be important allergens.New clinical and molecular insights advanced the knowledge and understanding of fish allergy in the last years. These findings will be useful for the advancement of the IgE-based diagnosis but also for the management of fish allergies consisting of advice and treatment of fish-allergic patients.

Characterisation of purified parvalbumin from five fish species and nucleotide sequencing of this major allergen from Pacific pilchard, Sardinops sagax.

Science.gov (United States)

Beale, Janine E; Jeebhay, Mohamed F; Lopata, Andreas L

2009-09-01

IgE-mediated allergic reaction to seafood is a common cause of food allergy including anaphylactic reactions. Parvalbumin, the major fish allergen, has been shown to display IgE cross-reactivity among fish species consumed predominantly in Europe and the Far East. However, cross-reactivity studies of parvalbumin from fish species widely consumed in the Southern hemisphere are limited as is data relating to immunological and molecular characterisation. In this study, antigenic cross-reactivity and the presence of oligomers and isomers of parvalbumin from five highly consumed fish species in Southern Africa were assessed by immunoblotting using purified parvalbumin and crude fish extracts. Pilchard (Sardinops sagax) parvalbumin was found to display the strongest IgE reactivity among 10 fish-allergic consumers. The cDNA sequence of the beta-form of pilchard parvalbumin was determined and designated Sar sa 1.0101 (accession number FM177701 EMBL/GenBank/DDBJ databases). Oligomeric forms of parvalbumin were observed in all fish species using a monoclonal anti-parvalbumin antibody and subject's sera. Isoforms varied between approximately 10-13 kDa. A highly cross-reactive allergenic isoform of parvalbumin was identified and sequenced, providing a successful primary step towards the generation of a recombinant form that could be used for diagnostic and potential therapeutic use in allergic individuals.

Parvalbumin in fish skin-derived gelatin: Is there a risk for fish allergic consumers?

NARCIS (Netherlands)

Koppelman, S.J.; Nordlee, J.A.; Lee, P.W.; Happe, R.P.; Hessing, M.; Norland, R.; Manning, T.; Deschene, R.; Jong, G.A.H.de; Taylor, S.L.

2012-01-01

The major allergen parvalbumin was purified from cod muscle tissues, and polyclonal antibodies were raised towards it. The antibodies were tested for specificity and an enzyme-linked immunosorbent assay (ELISA) was developed using these antibodies. The ELISA was applied to measure parvalbumin in cod

Chemical basis of prey recognition in thamnophiine snakes: the unexpected new roles of parvalbumins.

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Maïté Smargiassi

Full Text Available Detecting and locating prey are key to predatory success within trophic chains. Predators use various signals through specialized visual, olfactory, auditory or tactile sensory systems to pinpoint their prey. Snakes chemically sense their prey through a highly developed auxiliary olfactory sense organ, the vomeronasal organ (VNO. In natricine snakes that are able to feed on land and water, the VNO plays a critical role in predatory behavior by detecting cues, known as vomodors, which are produced by their potential prey. However, the chemical nature of these cues remains unclear. Recently, we demonstrated that specific proteins-parvalbumins-present in the cutaneous mucus of the common frog (Rana temporaria may be natural chemoattractive proteins for these snakes. Here, we show that parvalbumins and parvalbumin-like proteins, which are mainly intracellular, are physiologically present in the epidermal mucous cells and mucus of several frog and fish genera from both fresh and salt water. These proteins are located in many tissues and function as Ca(2+ buffers. In addition, we clarified the intrinsic role of parvalbumins present in the cutaneous mucus of amphibians and fishes. We demonstrate that these Ca(2+-binding proteins participate in innate bacterial defense mechanisms by means of calcium chelation. We show that these parvalbumins are chemoattractive for three different thamnophiine snakes, suggesting that these chemicals play a key role in their prey-recognition mechanism. Therefore, we suggest that recognition of parvalbumin-like proteins or other calcium-binding proteins by the VNO could be a generalized prey-recognition process in snakes. Detecting innate prey defense mechanism compounds may have driven the evolution of this predator-prey interaction.

Parvalbumin and calbindin immunoreactivity in the cerebral cortex of the hedgehog (Erinaceus europaeus).

Science.gov (United States)

Ferrer, I; Zujar, M J; Admella, C; Alcantara, S

1992-01-01

To investigate the morphology and distribution of nonpyramidal neurons in the brain of insectivores, parvalbumin and calbindin 28 kDa immunoreactivity was examined in the cerebral cortex of the hedgehog (Erinaceus europaeus). Parvalbumin-immunoreactive cells were found in all layers of the isocortex, but in contrast to other mammals, a laminar organisation or specific regional distribution was not seen. Characteristic parvalbumin-immunoreactive neurons were multipolar cells with large ascending and descending dendrites extending throughout several layers. Calbindin-immunoreactive neurons were similar to those found in other species, although appearing in smaller numbers than in the cerebral cortex of more advanced mammals. The morphology and distribution of parvalbumin- and calbindin-immunoreactive cells in the piriform and entorhinal cortices were similar in hedgehogs and rodents. Parvalbumin-immunoreactive cells in the hippocampal complex were pyramidal-like and bitufted neurons, which were mainly found in the stratum oriens and stratum pyramidale of the hippocampus, and in the stratum moleculare and hilus of the fascia dentata. Heavily stained cells were found in the deep part of the stratum granulare. Intense calbindin immunoreactivity occurred mainly in the granule cell and molecular layers of the dentate gyrus and in the mossy fibre layer. The most outstanding feature in the hippocampal complex of the hedgehog was the extension of calbindin immunoreactivity to CA1 field of the hippocampus, suggesting, in agreement with other reports, that mossy fibres can establish synaptic contacts throughout the pyramidal cell layer. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:1452472

Monosensitivity to pangasius and tilapia caused by allergens other than parvalbumin.

Science.gov (United States)

Ebo, D G; Kuehn, A; Bridts, C H; Hilger, C; Hentges, F; Stevens, W J

2010-01-01

Fish allergy is one of the most common food allergies in populations where fish is a major part of the diet. Most fish-allergic patients react to the panallergen parvalbumin present in multiple fish species. Our aim was to investigate the clinical case of a patient with oral allergy syndrome to pangasius and Nile tilapia but tolerance of other fish and seafood. The temporal relationship between fish consumption and allergic symptoms, the positive skin prick tests, and the basophil activation test results for both fish species strongly supported the diagnosis of an immunoglobulin (Ig) E-mediated allergy. This was confirmed by the detection of specific IgE to 18-kDa and 45-kDa proteins in immunoblot analysis. Notably, the patient was not sensitized to parvalbumin, as shown by enzyme-linked immunosorbent assay using purified allergens. Cross-reactivity between fish species can result from sensitization to allergens other than parvalbumin. This case report emphasizes the applications of flow cytometry-assisted analysis in the diagnosis of food allergy.

Fish β-parvalbumin acquires allergenic properties by amyloid assembly.

Science.gov (United States)

Martínez, Javier; Sánchez, Rosa; Castellanos, Milagros; Fernández-Escamilla, Ana M; Vázquez-Cortés, Sonia; Fernández-Rivas, Montserrat; Gasset, María

2015-01-01

Amyloids are highly cross-β-sheet-rich aggregated states that confer protease resistance, membrane activity and multivalence properties to proteins, all essential features for the undesired preservation of food proteins transiting the gastrointestinal tract and causing type I allergy. Amyloid propensity of β-parvalbumin, the major fish allergen, was theoretically analysed and assayed under gastrointestinal-relevant conditions using the binding of thioflavin T, the formation of sodium dodecyl sulphate- (SDS-) resistant aggregates, circular dichroism spectroscopy and atomic force microscopy fibril imaging. Impact of amyloid aggregates on allergenicity was assessed with dot blot. Sequences of β-parvalbumin from species with commercial value contain several adhesive hexapeptides capable of driving amyloid formation. Using Atlantic cod β-parvalbumin (rGad m 1) displaying high IgE cross-reactivity, we found that formation of amyloid fibres under simulated gastrointestinal conditions accounts for the resistance to acid and neutral proteases, for the presence of membrane active species under gastrointestinal relevant conditions and for the IgE-recognition in the sera of allergic patients. Incorporation of the anti-amyloid compound epigallocatechin gallate prevents rGad m 1 fibrillation, facilitates its protease digestion and impairs its recognition by IgE. the formation of amyloid by rGad m 1 explains its degradation resistance, its facilitated passage across the intestinal epithelial barrier and its epitope architecture as allergen.

Altered gamma oscillations during pregnancy through loss of δ subunit-containing GABAA receptors on parvalbumin interneurons

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Isabella eFerando

2013-09-01

Full Text Available Gamma (γ oscillations (30-120 Hz, an emergent property of neuronal networks, correlate with memory, cognition and encoding. In the hippocampal CA3 region, locally generated γ oscillations emerge through feedback between inhibitory parvalbumin-positive basket cells (PV+BCs and the principal (pyramidal cells. PV+BCs express δ-subunit-containing GABAARs (-GABAARs and NMDA receptors (NMDA-Rs that balance the frequency of γ oscillations. Neuroactive steroids (NS, such as the progesterone-derived (3α,5α-3-hydroxy-pregnan-20-one (allopregnanolone; ALLO, modulate the expression of δ-GABAARs and the tonic conductance they mediate. Pregnancy produces large increases in ALLO and brain-region-specific homeostatic changes in δ-GABAARs expression. Here we show that in CA3, where most PV+ interneurons (INs express δ-GABAARs, expression of δ-GABAARs on INs diminishes during pregnancy, but reverts to control levels within 48 hours postpartum. These anatomical findings were corroborated by a pregnancy-related increase in the frequency of kainate-induced CA3 γ oscillations in vitro that could be countered by the NMDA-R antagonists D-AP5 and PPDA. Mimicking the typical hormonal conditions during pregnancy by supplementing 100 nM ALLO lowered the γ frequencies to levels found in virgin or postpartum mice. Our findings show that states of altered NS levels (e.g., pregnancy may provoke perturbations in γ oscillatory activity through direct effects on the GABAergic system, and underscore the importance of δ-GABAARs homeostatic plasticity in maintaining constant network output despite large hormonal changes. Inaccurate coupling of NS levels to δ-GABAAR expression may facilitate abnormal neurological and psychiatric conditions such as epilepsy, post-partum depression, and post-partum psychosis, thus providing insights into potential new treatments.

A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.

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Merima Bublin

Full Text Available Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1, carp (Cyp c 1 and rainbow trout (Onc m 1 parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.

Science.gov (United States)

Bublin, Merima; Kostadinova, Maria; Fuchs, Julian E; Ackerbauer, Daniela; Moraes, Adolfo H; Almeida, Fabio C L; Lengger, Nina; Hafner, Christine; Ebner, Christof; Radauer, Christian; Liedl, Klaus R; Valente, Ana Paula; Breiteneder, Heimo

2015-01-01

Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

Characterization of parvalbumin, the major allergen in Alaska pollack, and comparison with codfish Allergen M.

Science.gov (United States)

Van Do, Thien; Hordvik, Ivar; Endresen, Curt; Elsayed, Said

2005-02-01

Increased fish consumption has led to frequent reporting of fish allergy and adverse reactions. Alaska pollack (Theragra chalcogramma) is a globally important commercial fish species, belonging to the Gadidae family. This family of fish also includes cod whose parvalbumin, Allergen M (Gad c 1), has been thoroughly studied and considered as a reference to sensitization in fish allergy. In the present study, parvalbumin from Alaska pollack, designated The c 1, was purified by use of anion exchange chromatography. To demonstrate the homogeneity of the purified protein, reverse phase high performance liquid chromatography was performed and showed two distinct fractions which had similar IgG and IgE binding capacities. Accordingly, cDNA cloning revealed two isotypic parvalbumin transcripts in pollack muscle. Recombinant parvalbumins of pollack exhibited low IgG and IgE binding capacities, in contrast to the native counterparts, which were almost as potent as cod Gad c 1. The allergenicity of The c 1 was assayed by ELISA inhibition, and compared to cod, the concentration required for obtaining 50% ELISA inhibition (C 50%) was only 18% higher for The c 1.

[Calbindin and parvalbumin distribution in spinal cord of normal and rabies-infected mice].

Science.gov (United States)

Monroy-Gómez, Jeison; Torres-Fernández, Orlando

2013-01-01

Rabies is a fatal infectious disease of the nervous system; however, the knowledge about the pathogenic neural mechanisms in rabies is scarce. In addition, there are few studies of rabies pathology of the spinal cord. To study the distribution of calcium binding proteins calbindin and parvalbumin and assessing the effect of rabies virus infection on their expression in the spinal cord of mice. MATERIALES Y METHODS: Mice were inoculated with rabies virus, by intracerebral or intramuscular route. The spinal cord was extracted to perform some crosscuts which were treated by immunohistochemistry with monoclonal antibodies to reveal the presence of the two proteins in normal and rabies infected mice. We did qualitative and quantitative analyses of the immunoreactivity of the two proteins. Calbindin and parvalbumin showed differential distribution in Rexed laminae. Rabies infection produced a decrease in the expression of calbindin. On the contrary, the infection caused an increased expression of parvalbumin. The effect of rabies infection on the two proteins expression was similar when comparing both routes of inoculation. The differential effect of rabies virus infection on the expression of calbindin and parvalbumin in the spinal cord of mice was similar to that previously reported for brain areas. This result suggests uniformity in the response to rabies infection throughout the central nervous system. This is an important contribution to the understanding of the pathogenesis of rabies.

Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells.

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Michelle Barbi de Moura

Full Text Available SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.

Severe IgE-mediated anaphylaxis following consumption of fried frog legs: definition of alpha-parvalbumin as the allergen in cause.

Science.gov (United States)

Hilger, C; Grigioni, F; Thill, L; Mertens, L; Hentges, F

2002-11-01

IgE-mediated allergic reactions to bullfrog and edible frog have been reported. The implicated allergens have not been defined so far. The frog material and the patient's serum from a case of severe food-induced anaphylaxis were used to define the implicated allergen at the protein and DNA level. Immunoblotting techniques and N-terminal protein microsequencing were used to define the allergen recognized by the patient's serum. Back translation from the identified protein sequence was used to design degenerated primers to amplify the allergen's cDNA by polymerase chain reaction (PCR). We defined the nucleotide sequence of the allergen from the frog of Indonesian origin that was consumed by the patient, and the homologous cDNA from Rana esculenta. Protein microsequencing revealed that the implicated frog allergen belonged to the parvalbumin family. cDNAs coding for alpha- and beta-parvalbumin of R. esculenta and Rana species were cloned. Recombinant proteins were expressed in Escherichia coli. The patient's serum IgE antibodies recognized parvalbumin prepared from frog muscle and recombinant alpha-parvalbumin from R. species but not from R. esculenta. Recombinant beta-parvalbumin was not recognized by the IgE antibodies. This work defines at the protein and DNA levels alpha-parvalbumin as the allergen implicated in a case of IgE-mediated anaphylaxis to frog muscle. It also shows that a protein belonging to the parvalbumin family is implicated in type I allergies outside the fish species.

Purification, biochemical, and immunological characterisation of a major food allergen: different immunoglobulin E recognition of the apo- and calcium-bound forms of carp parvalbumin.

Science.gov (United States)

Bugajska-Schretter, A; Grote, M; Vangelista, L; Valent, P; Sperr, W R; Rumpold, H; Pastore, A; Reichelt, R; Valenta, R; Spitzauer, S

2000-05-01

Almost 4% of the population suffer from food allergy which is an adverse reaction to food with an underlying immunological mechanism. To characterise one of the most frequent IgE defined food allergens, fish parvalbumin. Tissue and subcellular distribution of carp parvalbumin was analysed by immunogold electron microscopy and cell fractionation. Parvalbumin was purified to homogeneity, analysed by mass spectrometry and circular dichroism (CD) spectroscopy, and its allergenic activity was analysed by IgE binding and basophil histamine release tests. The isoelectric point (pI) 4.7 form of carp parvalbumin, a three EF-hand calcium-binding protein, was purified to homogeneity. CD analysis revealed a remarkable stability and refolding capacity of calcium-bound parvalbumin. This may explain why parvalbumin, despite cooking and exposure to the gastrointestinal tract, can sensitise patients. Purified parvalbumin reacted with IgE of more than 95% of individuals allergic to fish, induced dose-dependent basophil histamine release and contained, on average, 83% of the IgE epitopes present in other fish species. Calcium depletion reduced the IgE binding capacity of parvalbumin which, according to CD analysis, may be due to conformation-dependent IgE recognition. Purified carp parvalbumin represents an important cross reactive food allergen. It can be used for in vitro and in vivo diagnosis of fish-induced food allergy. Our finding that the apo-form of parvalbumin had a greatly reduced IgE binding capacity indicates that this form may be a candidate for safe immunotherapy of fish-related food allergy.

Prognostic and predictive values of EGFR overexpression and EGFR copy number alteration in HER2-positive breast cancer.

Science.gov (United States)

Lee, H J; Seo, A N; Kim, E J; Jang, M H; Kim, Y J; Kim, J H; Kim, S-W; Ryu, H S; Park, I A; Im, S-A; Gong, G; Jung, K H; Kim, H J; Park, S Y

2015-01-06

Epidermal growth factor receptor (EGFR) is overexpressed in a subset of human epidermal growth factor receptor 2 (HER2)-positive breast cancers, and coexpression of HER2 and EGFR has been reported to be associated with poor clinical outcome. Moreover, interaction between HER2 and EGFR has been suggested to be a possible basis for trastuzumab resistance. We analysed the clinical significance of EGFR overexpression and EGFR gene copy number alterations in 242 HER2-positive primary breast cancers. In addition, we examined the correlations between EGFR overexpression, trastuzumab response and clinical outcome in 447 primary, and 112 metastatic HER2-positive breast cancer patients treated by trastuzumab. Of the 242 primary cases, the level of EGFR overexpression was 2+ in 12.7% and 3+ in 11.8%. High EGFR gene copy number was detected in 10.3%. Epidermal growth factor receptor overexpression was associated with hormone receptor negativity and high Ki-67 proliferation index. In survival analyses, EGFR overexpression, but not high EGFR copy number, was associated with poor disease-free survival in all patients, and in the subgroup not receiving adjuvant trastuzumab. In 447 HER2-positive primary breast cancer patients treated with adjuvant trastuzumab, EGFR overexpression was also an independent poor prognostic factor. However, EGFR overexpression was not associated with trastuzumab response, progression-free survival or overall survival in the metastatic setting. Epidermal growth factor receptor overexpression, but not high EGFR copy number, is a poor prognostic factor in HER2-positive primary breast cancer. Epidermal growth factor receptor overexpression is a predictive factor for trastuzumab response in HER2-positive primary breast cancer, but not in metastatic breast cancer.

The opposite effects of nandrolone decanoate and exercise on anxiety levels in rats may involve alterations in hippocampal parvalbumin-positive interneurons.

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Dragica Selakovic

Full Text Available The aim of this study was to evaluate the behavioral effects of chronic (six weeks nandrolone decanoate (ND, 20 mg/kg, s.c., weekly in single dose administration (in order to mimic heavy human abuse, and exercise (swimming protocol of 60 minutes a day, five days in a row/two days break, applied alone and simultaneously with ND, in male rats (n = 40. Also, we evaluated the effects of those protocols on hippocampal parvalbumin (PV content and the possible connection between the alterations in certain parts of hippocampal GABAergic system and behavioral patterns. Both ND and exercise protocols induced increase in testosterone, dihydrotestosterone and estradiol blood levels. Our results confirmed anxiogenic effects of ND observed in open field (OF test (decrease in the locomotor activity, as well as in frequency and cumulative duration in the centre zone and in elevated plus maze (EPM test (decrease in frequency and cumulative duration in open arms, and total exploratory activity, that were accompanied with a mild decrease in the number of PV interneurons in hippocampus. Chronic exercise protocol induced significant increase in hippocampal PV neurons (dentate gyrus and CA1 region, followed by anxiolytic-like behavioral changes, observed in both OF and EPM (increase in all estimated parameters, and in evoked beam-walking test (increase in time to cross the beam, compared to ND treated animals. The applied dose of ND was sufficient to attenuate beneficial effects of exercise in rats by means of decreased exercise-induced anxiolytic effect, as well as to reverse exercise-induced augmentation in number of PV immunoreactive neurons in hippocampus. Our results implicate the possibility that alterations in hippocampal PV interneurons (i.e. GABAergic system may be involved in modulation of anxiety level induced by ND abuse and/or extended exercise protocols.

The opposite effects of nandrolone decanoate and exercise on anxiety levels in rats may involve alterations in hippocampal parvalbumin-positive interneurons.

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Selakovic, Dragica; Joksimovic, Jovana; Zaletel, Ivan; Puskas, Nela; Matovic, Milovan; Rosic, Gvozden

2017-01-01

The aim of this study was to evaluate the behavioral effects of chronic (six weeks) nandrolone decanoate (ND, 20 mg/kg, s.c., weekly in single dose) administration (in order to mimic heavy human abuse), and exercise (swimming protocol of 60 minutes a day, five days in a row/two days break), applied alone and simultaneously with ND, in male rats (n = 40). Also, we evaluated the effects of those protocols on hippocampal parvalbumin (PV) content and the possible connection between the alterations in certain parts of hippocampal GABAergic system and behavioral patterns. Both ND and exercise protocols induced increase in testosterone, dihydrotestosterone and estradiol blood levels. Our results confirmed anxiogenic effects of ND observed in open field (OF) test (decrease in the locomotor activity, as well as in frequency and cumulative duration in the centre zone) and in elevated plus maze (EPM) test (decrease in frequency and cumulative duration in open arms, and total exploratory activity), that were accompanied with a mild decrease in the number of PV interneurons in hippocampus. Chronic exercise protocol induced significant increase in hippocampal PV neurons (dentate gyrus and CA1 region), followed by anxiolytic-like behavioral changes, observed in both OF and EPM (increase in all estimated parameters), and in evoked beam-walking test (increase in time to cross the beam), compared to ND treated animals. The applied dose of ND was sufficient to attenuate beneficial effects of exercise in rats by means of decreased exercise-induced anxiolytic effect, as well as to reverse exercise-induced augmentation in number of PV immunoreactive neurons in hippocampus. Our results implicate the possibility that alterations in hippocampal PV interneurons (i.e. GABAergic system) may be involved in modulation of anxiety level induced by ND abuse and/or extended exercise protocols.

Overexpression of plastidial thioredoxins f and m differentially alters photosynthetic activity and response to oxidative stress in tobacco plants

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Pascal eREY

2013-10-01

Full Text Available Plants display a remarkable diversity of thioredoxins (Trxs, reductases controlling the thiol redox status of proteins. The physiological function of many of them remains elusive, particularly for plastidial Trxs f and m, which are presumed based on biochemical data to regulate photosynthetic reactions and carbon metabolism. Recent reports revealed that Trxs f and m participate in vivo in the control of starch metabolism and cyclic photosynthetic electron transfer around photosystem I, respectively. To further delineate their in planta function, we compared the photosynthetic characteristics, the level and/or activity of various Trx targets and the responses to oxidative stress in transplastomic tobacco plants overexpressing either Trx f or Trx m. We found that plants overexpressing Trx m specifically exhibit altered growth, reduced chlorophyll content, impaired photosynthetic linear electron transfer and decreased pools of glutathione and ascorbate. In both transplastomic lines, activities of two enzymes involved in carbon metabolism, NADP-malate dehydrogenase and NADP-glyceraldehyde-3-phosphate dehydrogenase are markedly and similarly altered. In contrast, plants overexpressing Trx m specifically display increased capacity for methionine sulfoxide reductases, enzymes repairing damaged proteins by regenerating methionine from oxidized methionine. Finally, we also observed that transplastomic plants exhibit distinct responses when exposed to oxidative stress conditions generated by methyl viologen or exposure to high light combined with low temperature, the plants overexpressing Trx m being notably more tolerant than Wt and those overexpressing Trx f. Altogether, these data indicate that Trxs f and m fulfill distinct physiological functions. They prompt us to propose that the m type is involved in key processes linking photosynthetic activity, redox homeostasis and antioxidant mechanisms in the chloroplast.

Overexpression of AtLOV1 in Switchgrass alters plant architecture, lignin content, and flowering time.

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Bin Xu

Full Text Available BACKGROUND: Switchgrass (Panicum virgatum L. is a prime candidate crop for biofuel feedstock production in the United States. As it is a self-incompatible polyploid perennial species, breeding elite and stable switchgrass cultivars with traditional breeding methods is very challenging. Translational genomics may contribute significantly to the genetic improvement of switchgrass, especially for the incorporation of elite traits that are absent in natural switchgrass populations. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we constitutively expressed an Arabidopsis NAC transcriptional factor gene, LONG VEGETATIVE PHASE ONE (AtLOV1, in switchgrass. Overexpression of AtLOV1 in switchgrass caused the plants to have a smaller leaf angle by changing the morphology and organization of epidermal cells in the leaf collar region. Also, overexpression of AtLOV1 altered the lignin content and the monolignol composition of cell walls, and caused delayed flowering time. Global gene-expression analysis of the transgenic plants revealed an array of responding genes with predicted functions in plant development, cell wall biosynthesis, and flowering. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first report of a single ectopically expressed transcription factor altering the leaf angle, cell wall composition, and flowering time of switchgrass, therefore demonstrating the potential advantage of translational genomics for the genetic improvement of this crop.

Two Cases of Occupational Contact Urticaria Caused by Percutaneous Sensitization to Parvalbumin

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Akiyo Sano

2015-08-01

Full Text Available Background: In recent years, it has been proposed that the primary mechanism for the development of food allergies is percutaneous sensitization. Since 2010, in Japan, the number of immediate-type wheat allergy due to hydrolyzed wheat protein has dramatically increased among those who have been using soap containing hydrolyzed wheat. This incidence supports the hypothesis that food allergens arise through percutaneous sensitization. Clinical Summary: A 25-year-old man (case 1 and an 18-year-old girl (case 2 with atopic dermatitis visited our Department because of food allergy and hand eczema. After starting their work with fish, severe itchy eczema appeared on their hands. They subsequently started to experience oral allergic symptoms, intraoral itchiness and dyspnea after eating fish. Specific IgE antibodies were detected for many fishes, and skin prick tests showed positive reactions for a variety of fishes in both cases. Furthermore, the fluorescence intensities of specific IgE antibodies against parvalbumin from various types of fish in microarray immunoassay analysis showed positive reactions. We diagnosed them as contact urticaria caused by percutaneous sensitization to parvalbumin through job-related physical contact with fish. Conclusion: The patients' histories and findings indicate the possibility of percutaneous sensitization through occupational exposure to parvalbumin, leading to food allergy.

Two Cases of Occupational Contact Urticaria Caused by Percutaneous Sensitization to Parvalbumin.

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Sano, Akiyo; Yagami, Akiko; Suzuki, Kayoko; Iwata, Yohei; Kobayashi, Tsukane; Arima, Masaru; Kondo, Yasuto; Yoshikawa, Tetsushi; Matsunaga, Kayoko

2015-01-01

In recent years, it has been proposed that the primary mechanism for the development of food allergies is percutaneous sensitization. Since 2010, in Japan, the number of immediate-type wheat allergy due to hydrolyzed wheat protein has dramatically increased among those who have been using soap containing hydrolyzed wheat. This incidence supports the hypothesis that food allergens arise through percutaneous sensitization. A 25-year-old man (case 1) and an 18-year-old girl (case 2) with atopic dermatitis visited our Department because of food allergy and hand eczema. After starting their work with fish, severe itchy eczema appeared on their hands. They subsequently started to experience oral allergic symptoms, intraoral itchiness and dyspnea after eating fish. Specific IgE antibodies were detected for many fishes, and skin prick tests showed positive reactions for a variety of fishes in both cases. Furthermore, the fluorescence intensities of specific IgE antibodies against parvalbumin from various types of fish in microarray immunoassay analysis showed positive reactions. We diagnosed them as contact urticaria caused by percutaneous sensitization to parvalbumin through job-related physical contact with fish. The patients' histories and findings indicate the possibility of percutaneous sensitization through occupational exposure to parvalbumin, leading to food allergy.

Functional characteristics of parvalbumin- and cholecystokinin-expressing basket cells.

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Bartos, Marlene; Elgueta, Claudio

2012-02-15

Cortical neuronal network operations depend critically on the recruitment of GABAergic interneurons and the properties of their inhibitory output signals. Recent evidence indicates a marked difference in the signalling properties of two major types of perisomatic inhibitory interneurons, the parvalbumin- and the cholecystokinin-containing basket cells. Parvalbumin-expressing basket cells are rapidly recruited by excitatory synaptic inputs, generate high-frequency trains of action potentials, discharge single action potentials phase-locked to fast network oscillations and provide fast, stable and timed inhibitory output onto their target cells. In contrast, cholecystokinin-containing basket cells are recruited in a less reliable manner, discharge at moderate frequencies with single action potentials weakly coupled to the phases of fast network oscillations and generate an asynchronous, fluctuating and less timed inhibitory output. These signalling modes are based on cell type-dependent differences in the functional and plastic properties of excitatory input synapses, integrative qualities and in the kinetics and dynamics of inhibitory output synapses. Thus, the two perisomatic inhibitory interneuron types operate with different speed and precision and may therefore contribute differently to the operations of neuronal networks.

Overexpression of antioxidant enzymes in diaphragm muscle does not alter contraction-induced fatigue or recovery

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McClung, Joseph M.; DeRuisseau, Keith C.; Whidden, Melissa A.; Van Remmen, Holly; Richardson, Arlan; Song, Wook; Vrabas, Ioannis S.; Powers, Scott K.

2010-01-01

contractile dysfunction. Finally, the transgenic overexpression of independent endogenous antioxidants alters diaphragm skeletal muscle morphology, and these changes may also contribute to the diminished specific force production observed in these animals. PMID:19783618

Solution structure of the major fish allergen parvalbumin Sco j 1 derived from the Pacific mackerel

OpenAIRE

Kumeta, Hiroyuki; Nakayama, Haruka; Ogura, Kenji

2017-01-01

Although fish is an important part of the human diet, it is also a common source of food allergy. The major allergen in fish is parvalbumin, a well-conserved Ca2+-binding protein found in the white muscle of many fish species. Here, we studied the solution structure of the parvalbumin Sco j 1, derived from the Pacific mackerel, using nuclear magnetic resonance spectroscopy. We mapped the IgE-binding epitope proposed in a recent study onto the present structure. Interestingly, three of four re...

Recombinant carp parvalbumin, the major cross-reactive fish allergen: a tool for diagnosis and therapy of fish allergy.

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Swoboda, Ines; Bugajska-Schretter, Agnes; Verdino, Petra; Keller, Walter; Sperr, Wolfgang R; Valent, Peter; Valenta, Rudolf; Spitzauer, Susanne

2002-05-01

IgE-mediated reactions to fish allergens represent one of the most frequent causes of food allergy. We have constructed an expression cDNA library from carp (Cyprinus carpio) muscle in phage lambda gt11 and used serum IgE from a fish allergic patient to isolate 33 cDNA clones that coded for two parvalbumin isoforms (Cyp c 1.01 and Cyp c 1.02) with comparable IgE binding capacities. Both isoforms represented calcium-binding proteins that belonged to the beta-lineage of parvalbumins. The Cyp c 1.01 cDNA was overexpressed in Escherichia coli, and rCyp c 1.01 was purified to homogeneity. Circular dichroism analysis and mass spectroscopy showed that rCyp c 1.01 represented a folded protein with mainly alpha-helical secondary structure and a molecular mass of 11,416 Da, respectively. rCyp c 1.01 reacted with IgE from all fish-allergic patients tested (n = 60), induced specific and dose-dependent basophil histamine release, and contained most of the IgE epitopes (70%) present in natural allergen extracts from cod, tuna, and salmon. Therefore, it may be used to identify patients suffering from IgE-mediated fish allergy. The therapeutic potential of rCyp c 1.01 is indicated by our findings that rabbit Abs raised against rCyp c 1.01 inhibited the binding of IgE (n = 25) in fish-allergic patients to rCyp c 1.01 between 35 and 97% (84% mean inhibition) and that depletion of calcium strongly reduced IgE recognition of rCyp c 1.01. The latter results suggest that it will be possible to develop strategies for immunotherapy for fish allergy that are based on calcium-free hypoallergenic rCyp c 1.01 derivatives.

Convergence of genetic and environmental factors on parvalbumin-positive interneurons in schizophrenia

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Zhihong eJiang

2013-09-01

Full Text Available Schizophrenia etiology is thought to involve an interaction between genetic and environmental factors during postnatal brain development. However, there is a fundamental gap in our understanding of the molecular mechanisms by which environmental factors interact with genetic susceptibility to trigger symptom onset and disease progression. In this review, we summarize the most recent findings implicating oxidative stress as one mechanism by which environmental insults, especially early life social stress, impact the development of schizophrenia. Based on a review of the literature and the results of our own animal model, we suggest that environmental stressors such as social isolation render parvalbumin-positive interneurons vulnerable to oxidative stress. We previously reported that social isolation stress exacerbates many of the schizophrenia-like phenotypes seen in a conditional genetic mouse model of schizophrenia in which NMDARs are selectively ablated in half of cortical and hippocampal interneurons during early postnatal development (Belforte et al., 2010. We have since revealed that this social isolation-induced effect is caused by impairments in the antioxidant defense capacity in the parvalbumin-positive interneurons in which NMDARs are ablated. We propose that this effect is mediated by the down-regulation of PGC-1α, a master regulator of mitochondrial energy metabolism and anti-oxidant defense, following the deletion of NMDARs (Jiang et al, 2013. Other potential molecular mechanisms underlying redox dysfunction upon gene and environmental interaction will be discussed, with a focus on the unique properties of parvalbumin-positive interneurons.

Purification and characterization of parvalbumin isotypes from grass carp (Ctenopharyngodon idella).

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Li, Zheng; You, Juan; Luo, Yongkang; Wu, Jianping

2014-07-02

The prevalence of fish allergy is rapidly increasing because of a growing fish consumption driven mainly by a positive image of the fish and health relationship. The purpose of this study was to characterize parvalbumin isotypes from grass carp (Ctenopharyngodon idella), one of the most frequently consumed freshwater fish in China. Three parvalbumin isotypes were purified using consecutive gel filtration and reverse-phase chromatography and denoted as PVI, PVII, and PVIII. The molecular weights of the isotypes were determined to be 11.968, 11.430, and 11.512 kDa, respectively. PVI showed 74% matched amino acids sequence with PV isotype 4a from Danio rerio, while PVII and PVIII showed 46% matched amino acids sequence with PV isotypes from Hypophthalmichthys molitrix. PVII is the dominant allergen, but it was liable to gastrointestinal enzymes as PVIII; however, PVI was resistant to pepsin digestion. A further study is to characterize the epitopes of PVII, the dominant allergen.

Identification of enolases and aldolases as important fish allergens in cod, salmon and tuna: component resolved diagnosis using parvalbumin and the new allergens.

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Kuehn, A; Hilger, C; Lehners-Weber, C; Codreanu-Morel, F; Morisset, M; Metz-Favre, C; Pauli, G; de Blay, F; Revets, D; Muller, C P; Vogel, L; Vieths, S; Hentges, F

2013-07-01

The majority of fish-allergic patients are sensitized to parvalbumin, known to be the cause of important IgE cross-reactivity among fish species. Little is known about the importance of fish allergens other than parvalbumin. The aim of this study was to characterize hitherto undefined fish allergens in three commonly consumed fish species, cod, salmon and tuna, and to evaluate their importance for in vitro IgE-diagnosis in addition to parvalbumin and fish gelatin. Sixty-two patients were diagnosed by clinical history, skin prick tests and specific IgE to fish extracts. Two new fish allergens from cod, salmon and tuna were identified by microsequencing. These proteins were characterized by immunoblot, ELISA and mediator release assay. Purified parvalbumin, enolase, aldolase and fish gelatin were used for quantification of specific IgE in ELISA. Parvalbumin and two other allergens of 50 and 40 kDa were detected in IgE-immunoblots of cod, salmon and tuna extracts by most patient sera. The 50 and 40 kDa proteins were identified as beta-enolase and fructose-bisphosphate aldolase A respectively. Both purified enzymes showed allergenic activity in the mediator release assay. Indeed, 72.6% of the patients were sensitized to parvalbumin, 20% of these had specific IgE to salmon parvalbumin only. IgE to enolases were found in 62.9% (0.5-95.0 kUA /L), to aldolases in 50.0% (0.4-26.0 kUA /L) and to fish gelatin in 19.3% (0.4-20.0 kUA /L) of the patients. Inter-species cross-reactivity, even though limited, was found for enolases and aldolases by IgE-inhibition ELISA. Fish enolase and aldolase have been identified as important new fish allergens. In fish allergy diagnosis, IgE to enolase and aldolase are especially relevant when IgE to parvalbumin are absent. © 2013 John Wiley & Sons Ltd.

Purification, biochemical, and immunological characterisation of a major food allergen: different immunoglobulin E recognition of the apo- and calcium-bound forms of carp parvalbumin

OpenAIRE

Bugajska-Schrette..., A; Grote, M; Vangelista, L; Valent, P; Sperr, W; Rumpold, H; Pastore, A; Reichelt, R; Valenta, R; Spitzauer, S

2000-01-01

BACKGROUND—Almost 4% of the population suffer from food allergy which is an adverse reaction to food with an underlying immunological mechanism.
AIMS—To characterise one of the most frequent IgE defined food allergens, fish parvalbumin.
METHODS—Tissue and subcellular distribution of carp parvalbumin was analysed by immunogold electron microscopy and cell fractionation. Parvalbumin was purified to homogeneity, analysed by mass spectrometry and circular dichroism (CD) spectroscopy, and its alle...

Alterations of cortical GABA neurons and network oscillations in schizophrenia.

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Gonzalez-Burgos, Guillermo; Hashimoto, Takanori; Lewis, David A

2010-08-01

The hypothesis that alterations of cortical inhibitory gamma-aminobutyric acid (GABA) neurons are a central element in the pathology of schizophrenia has emerged from a series of postmortem studies. How such abnormalities may contribute to the clinical features of schizophrenia has been substantially informed by a convergence with basic neuroscience studies revealing complex details of GABA neuron function in the healthy brain. Importantly, activity of the parvalbumin-containing class of GABA neurons has been linked to the production of cortical network oscillations. Furthermore, growing knowledge supports the concept that gamma band oscillations (30-80 Hz) are an essential mechanism for cortical information transmission and processing. Herein we review recent studies further indicating that inhibition from parvalbumin-positive GABA neurons is necessary to produce gamma oscillations in cortical circuits; provide an update on postmortem studies documenting that deficits in the expression of glutamic acid decarboxylase67, which accounts for most GABA synthesis in the cortex, are widely observed in schizophrenia; and describe studies using novel, noninvasive approaches directly assessing potential relations between alterations in GABA, oscillations, and cognitive function in schizophrenia.

Parvalbumin+ Neurons and Npas1+ Neurons Are Distinct Neuron Classes in the Mouse External Globus Pallidus.

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Hernández, Vivian M; Hegeman, Daniel J; Cui, Qiaoling; Kelver, Daniel A; Fiske, Michael P; Glajch, Kelly E; Pitt, Jason E; Huang, Tina Y; Justice, Nicholas J; Chan, C Savio

2015-08-26

Compelling evidence suggests that pathological activity of the external globus pallidus (GPe), a nucleus in the basal ganglia, contributes to the motor symptoms of a variety of movement disorders such as Parkinson's disease. Recent studies have challenged the idea that the GPe comprises a single, homogenous population of neurons that serves as a simple relay in the indirect pathway. However, we still lack a full understanding of the diversity of the neurons that make up the GPe. Specifically, a more precise classification scheme is needed to better describe the fundamental biology and function of different GPe neuron classes. To this end, we generated a novel multicistronic BAC (bacterial artificial chromosome) transgenic mouse line under the regulatory elements of the Npas1 gene. Using a combinatorial transgenic and immunohistochemical approach, we discovered that parvalbumin-expressing neurons and Npas1-expressing neurons in the GPe represent two nonoverlapping cell classes, amounting to 55% and 27% of the total GPe neuron population, respectively. These two genetically identified cell classes projected primarily to the subthalamic nucleus and to the striatum, respectively. Additionally, parvalbumin-expressing neurons and Npas1-expressing neurons were distinct in their autonomous and driven firing characteristics, their expression of intrinsic ion conductances, and their responsiveness to chronic 6-hydroxydopamine lesion. In summary, our data argue that parvalbumin-expressing neurons and Npas1-expressing neurons are two distinct functional classes of GPe neurons. This work revises our understanding of the GPe, and provides the foundation for future studies of its function and dysfunction. Until recently, the heterogeneity of the constituent neurons within the external globus pallidus (GPe) was not fully appreciated. We addressed this knowledge gap by discovering two principal GPe neuron classes, which were identified by their nonoverlapping expression of the

Parvalbumin+ Neurons and Npas1+ Neurons Are Distinct Neuron Classes in the Mouse External Globus Pallidus

Science.gov (United States)

Hernández, Vivian M.; Hegeman, Daniel J.; Cui, Qiaoling; Kelver, Daniel A.; Fiske, Michael P.; Glajch, Kelly E.; Pitt, Jason E.; Huang, Tina Y.; Justice, Nicholas J.

2015-01-01

Compelling evidence suggests that pathological activity of the external globus pallidus (GPe), a nucleus in the basal ganglia, contributes to the motor symptoms of a variety of movement disorders such as Parkinson's disease. Recent studies have challenged the idea that the GPe comprises a single, homogenous population of neurons that serves as a simple relay in the indirect pathway. However, we still lack a full understanding of the diversity of the neurons that make up the GPe. Specifically, a more precise classification scheme is needed to better describe the fundamental biology and function of different GPe neuron classes. To this end, we generated a novel multicistronic BAC (bacterial artificial chromosome) transgenic mouse line under the regulatory elements of the Npas1 gene. Using a combinatorial transgenic and immunohistochemical approach, we discovered that parvalbumin-expressing neurons and Npas1-expressing neurons in the GPe represent two nonoverlapping cell classes, amounting to 55% and 27% of the total GPe neuron population, respectively. These two genetically identified cell classes projected primarily to the subthalamic nucleus and to the striatum, respectively. Additionally, parvalbumin-expressing neurons and Npas1-expressing neurons were distinct in their autonomous and driven firing characteristics, their expression of intrinsic ion conductances, and their responsiveness to chronic 6-hydroxydopamine lesion. In summary, our data argue that parvalbumin-expressing neurons and Npas1-expressing neurons are two distinct functional classes of GPe neurons. This work revises our understanding of the GPe, and provides the foundation for future studies of its function and dysfunction. SIGNIFICANCE STATEMENT Until recently, the heterogeneity of the constituent neurons within the external globus pallidus (GPe) was not fully appreciated. We addressed this knowledge gap by discovering two principal GPe neuron classes, which were identified by their nonoverlapping

Advances on the molecular characterization, clinical relevance, and detection methods of Gadiform parvalbumin allergens.

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Fernandes, Telmo J R; Costa, Joana; Carrapatoso, Isabel; Oliveira, Maria Beatriz P P; Mafra, Isabel

2017-10-13

Gadiform order includes several fish families, from which Gadidae and Merlucciidae are part of, comprising the most commercially important and highly appreciated fish species, such as cod, pollock, haddock, and hake. Parvalbumins, classified as calcium-binding proteins, are considered the main components involved in the majority of fish allergies. Nine and thirteen parvalbumins were identified in different fish species from Gadidae and Merlucciidae families, respectively. This review intends to describe their molecular characterization and the clinical relevance, as well as the prevalence of fish allergy. In addition, the main protein- and DNA-based methods to detect fish allergens are fully reviewed owing to their importance in the safeguard of sensitized/allergic individuals.

Overexpression of NtWRKY50 Increases Resistance to Ralstonia solanacearum and Alters Salicylic Acid and Jasmonic Acid Production in Tobacco.

Science.gov (United States)

Liu, Qiuping; Liu, Ying; Tang, Yuanman; Chen, Juanni; Ding, Wei

2017-01-01

WRKY transcription factors (TFs) modulate plant responses to biotic and abiotic stresses. Here, we characterized a WRKY IIc TF, NtWRKY50, isolated from tobacco ( Nicotiana tabacum ) plants. The results showed that NtWRKY50 is a nuclear-localized protein and that its gene transcript is induced in tobacco when inoculated with the pathogenic bacterium Ralstonia solanacearum . Overexpression of NtWRKY50 enhanced bacterial resistance, which correlated with enhanced SA and JA/ET signaling genes. However, silencing of the NtWRKY50 gene had no obvious effects on plant disease resistance, implying functional redundancy of NtWRKY50 with other TFs. In addition, it was found that NtWRKY50 can be induced by various biotic or abiotic stresses, such as Potato virus Y, Rhizoctonia solani, Phytophthora parasitica , hydrogen peroxide, heat, cold, and wounding as well as the hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). Importantly, additional analysis suggests that NtWRKY50 overexpression markedly promotes SA levels but prevents pathogen-induced JA production. These data indicate that NtWRKY50 overexpression leads to altered SA and JA content, increased expression of defense-related genes and enhanced plant resistance to R. solanacearum. These probably due to increased activity of endogenous NtWRKY50 gene or could be gain-of-function phenotypes by altering the profile of genes affected by NtWRKY50 .

The 5-HT1A serotonin receptor is located on calbindin- and parvalbumin-containing neurons in the rat brain

DEFF Research Database (Denmark)

Aznar, Susana; Qian, Zhaoxia; Shah, Reshma

2003-01-01

distributed in the rat brain, with a particularly high density in the limbic system. The receptor's localization in the different neuronal subtypes, which may be of importance for understanding its role in neuronal circuitries, is, however, unknown. In this study we show by immunocytochemical double......-labeling techniques, that the 5-HT(1A) receptor is present on both pyramidal and principal cells, and calbindin- and parvalbumin-containing neurons, which generally define two different subtypes of interneurons. Moreover, semiquantitative analysis showed that the receptor's distribution in the different neuronal...... types varies between brain areas. In cortex, hippocampus, hypothalamus, and amygdala the receptor was located on both principal cells and calbindin- and parvalbumin-containing neurons. In septum and thalamus, the receptor was mostly present on calbindin- and parvalbumin-containing cells. Especially...

Overexpression of GRß in colonic mucosal cell line partly reflects altered gene expression in colonic mucosa of patients with inflammatory bowel disease.

Science.gov (United States)

Nagy, Zsolt; Acs, Bence; Butz, Henriett; Feldman, Karolina; Marta, Alexa; Szabo, Peter M; Baghy, Kornelia; Pazmany, Tamas; Racz, Karoly; Liko, Istvan; Patocs, Attila

2016-01-01

The glucocorticoid receptor (GR) plays a crucial role in inflammatory responses. GR has several isoforms, of which the most deeply studied are the GRα and GRß. Recently it has been suggested that in addition to its negative dominant effect on GRα, the GRß may have a GRα-independent transcriptional activity. The GRß isoform was found to be frequently overexpressed in various autoimmune diseases, including inflammatory bowel disease (IBD). In this study, we wished to test whether the gene expression profile found in a GRß overexpressing intestinal cell line (Caco-2GRß) might mimic the gene expression alterations found in patients with IBD. Whole genome microarray analysis was performed in both normal and GRß overexpressing Caco-2 cell lines with and without dexamethasone treatment. IBD-related genes were identified from a meta-analysis of 245 microarrays available in online microarray deposits performed on intestinal mucosa samples from patients with IBD and healthy individuals. The differentially expressed genes were further studied using in silico pathway analysis. Overexpression of GRß altered a large proportion of genes that were not regulated by dexamethasone suggesting that GRß may have a GRα-independent role in the regulation of gene expression. About 10% of genes differentially expressed in colonic mucosa samples from IBD patients compared to normal subjects were also detected in Caco-2 GRß intestinal cell line. Common genes are involved in cell adhesion and cell proliferation. Overexpression of GRß in intestinal cells may affect appropriate mucosal repair and intact barrier function. The proposed novel role of GRß in intestinal epithelium warrants further studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Overexpression of Dyrk1A is implicated in several cognitive, electrophysiological and neuromorphological alterations found in a mouse model of Down syndrome.

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Susana García-Cerro

Full Text Available Down syndrome (DS phenotypes result from the overexpression of several dosage-sensitive genes. The DYRK1A (dual-specificity tyrosine-(Y-phosphorylation regulated kinase 1A gene, which has been implicated in the behavioral and neuronal alterations that are characteristic of DS, plays a role in neuronal progenitor proliferation, neuronal differentiation and long-term potentiation (LTP mechanisms that contribute to the cognitive deficits found in DS. The purpose of this study was to evaluate the effect of Dyrk1A overexpression on the behavioral and cognitive alterations in the Ts65Dn (TS mouse model, which is the most commonly utilized mouse model of DS, as well as on several neuromorphological and electrophysiological properties proposed to underlie these deficits. In this study, we analyzed the phenotypic differences in the progeny obtained from crosses of TS females and heterozygous Dyrk1A (+/- male mice. Our results revealed that normalization of the Dyrk1A copy number in TS mice improved working and reference memory based on the Morris water maze and contextual conditioning based on the fear conditioning test and rescued hippocampal LTP. Concomitant with these functional improvements, normalization of the Dyrk1A expression level in TS mice restored the proliferation and differentiation of hippocampal cells in the adult dentate gyrus (DG and the density of GABAergic and glutamatergic synapse markers in the molecular layer of the hippocampus. However, normalization of the Dyrk1A gene dosage did not affect other structural (e.g., the density of mature hippocampal granule cells, the DG volume and the subgranular zone area or behavioral (i.e., hyperactivity/attention alterations found in the TS mouse. These results suggest that Dyrk1A overexpression is involved in some of the cognitive, electrophysiological and neuromorphological alterations, but not in the structural alterations found in DS, and suggest that pharmacological strategies targeting

Parvalbumin, a cross-reactive fish allergen, contains IgE-binding epitopes sensitive to periodate treatment and Ca2+ depletion.

Science.gov (United States)

Bugajska-Schretter, A; Elfman, L; Fuchs, T; Kapiotis, S; Rumpold, H; Valenta, R; Spitzauer, S

1998-01-01

Type I allergy to fish is a severe health problem in countries in which a large percentage of the population derive income from fishing. The aim of the study was to characterize cross-reactive IgE-binding components in six different fish species (cod, tuna, salmon, perch, carp, and eel). The effect of reducing extraction conditions, periodate treatment, and depletion of Ca2+ on binding of IgE to the allergens was investigated. Extracts were prepared under nonreducing and reducing conditions. IgE-binding components were characterized by IgE immunoblotting, and cross-reactive epitopes were studied by IgE-immunoblot inhibition experiments. To reveal calcium-sensitive or carbohydrate-containing epitopes, nitrocellulose-blotted extracts were exposed to ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and periodate. Sera from all patients allergic to fish (n = 30) displayed IgE reactivity to parvalbumin, a 12 kd protein present in fish extracts from six different species. Reducing extraction conditions had no effect on IgE binding to parvalbumins, whereas periodate treatment and depletion of protein-bound calcium led to a substantial reduction of IgE binding. Parvalbumins from six different species contained cross-reactive IgE epitopes. Parvalbumin represents a cross-reactive fish allergen. It contains IgE epitopes that are sensitive to periodate treatment and Ca2+-depletion.

Differential expression of parvalbumin interneurons in neonatal phencyclidine treated rats and socially isolated rats

DEFF Research Database (Denmark)

Kaalund, Sanne Simone; Riise, Jesper; Broberg, Brian

2013-01-01

of parvalbumin-positive interneurons (PV(+) interneurons). In this study we examined PV(+) expression in two rat models of cognitive dysfunction in schizophrenia, the environmental social isolation (SI) and pharmacological neonatal phencyclidine (neoPCP) models. Using a stereological method, the optical...

Altered cortical expression of GABA-related genes in schizophrenia: illness progression vs developmental disturbance.

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Hoftman, Gil D; Volk, David W; Bazmi, H Holly; Li, Siyu; Sampson, Allan R; Lewis, David A

2015-01-01

Schizophrenia is a neurodevelopmental disorder with altered expression of GABA-related genes in the prefrontal cortex (PFC). However, whether these gene expression abnormalities reflect disturbances in postnatal developmental processes before clinical onset or arise as a consequence of clinical illness remains unclear. Expression levels for 7 GABA-related transcripts (vesicular GABA transporter [vGAT], GABA membrane transporter [GAT1], GABAA receptor subunit α1 [GABRA1] [novel in human and monkey cohorts], glutamic acid decarboxylase 67 [GAD67], parvalbumin, calretinin, and somatostatin [previously reported in human cohort, but not in monkey cohort]) were quantified in the PFC from 42 matched pairs of schizophrenia and comparison subjects and from 49 rhesus monkeys ranging in age from 1 week postnatal to adulthood. Levels of vGAT and GABRA1, but not of GAT1, messenger RNAs (mRNAs) were lower in the PFC of the schizophrenia subjects. As previously reported, levels of GAD67, parvalbumin, and somatostatin, but not of calretinin, mRNAs were also lower in these subjects. Neither illness duration nor age accounted for the levels of the transcripts with altered expression in schizophrenia. In monkey PFC, developmental changes in expression levels of many of these transcripts were in the opposite direction of the changes observed in schizophrenia. For example, mRNA levels for vGAT, GABRA1, GAD67, and parvalbumin all increased with age. Together with published reports, these findings support the interpretation that the altered expression of GABA-related transcripts in schizophrenia reflects a blunting of normal postnatal development changes, but they cannot exclude a decline during the early stages of clinical illness. © The Author 2013. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Altered Fruit and Seed Development of Transgenic Rapeseed (Brassica napus Over-Expressing MicroRNA394.

Directory of Open Access Journals (Sweden)

Jian Bo Song

Full Text Available Fruit and seed development in plants is a complex biological process mainly involved in input and biosynthesis of many storage compounds such as proteins and oils. Although the basic biochemical pathways for production of the storage metabolites in plants are well characterized, their regulatory mechanisms are not fully understood. In this study, we functionally identified rapeseed (Brassica napus miR394 with its target gene Brassica napus leaf curling responsiveness (BnLCR to dissect a role of miR394 during the fruit and seed development. Transgenic rapeseed plants over-expressing miR394 under the control of the cauliflower mosaic virus 35S promoter were generated. miR394 over-expression plants exhibited a delayed flowering time and enlarged size of plants, leaf blade, pods and seed body, but developed seeds with higher contents of protein and glucosinolates (GLS and lower levels of oil accumulation as compared to wild-type. Over-expression of miR394 altered the fatty acid (FA composition by increasing several FA species such as C16:0 and C18:0 and unsaturated species of C20:1 and C22:1 but lowering C18:3. This change was accompanied by induction of genes coding for transcription factors of FA synthesis including leafy cotyledon1 (BnLEC1, BnLEC2, and FUSCA3 (FUS3. Because the phytohormone auxin plays a crucial role in fruit development and seed patterning, the DR5-GUS reporter was used for monitoring the auxin response in Arabidopsis siliques and demonstrated that the DR5 gene was strongly expressed. These results suggest that BnmiR394 is involved in rapeseed fruit and seed development.

Alterations in the 5 'untranslated region of the EPSPS gene influence EPSPS overexpression in glyphosate-resistant Eleusine indica.

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Zhang, Chun; Feng, Li; Tian, Xing-Shan

2018-04-26

The herbicide glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds, but the transcriptional regulation of this gene is poorly understood. The EPSPS gene was found to be significantly up-regulated following glyphosate treatment in a glyphosate- resistant Eleusine indica population from South China. To further investigate the regulation of EPSPS overexpression, the promoter of the EPSPS gene from this E. indica population was cloned and analyzed. Two upstream regulatory sequences, Epro-S (862 bp) and Epro-R (877 bp) of EPSPS were obtained from glyphosate-susceptible (S) and -resistant (R) E. indica plants respectively by HiTAIL-PCR. The Epro-S and Epro-R sequences were 99% homologous, except for the two insertions (3 bp and12 bp) in the R sequence. The 12-base insertion of the Epro-R sequence was located in the 5'-UTR-Py-rich stretch element. The promoter activity tests showed that the 12-base insertion resulted in significant enhancement of the Epro-R promoter activity, whereas the 3-base insertion had little effect on Epro-R promoter activity. Alterations in the 5'-UTR-Py-rich stretch element of EPSPS are responsible for glyphosate induced EPSPS overexpression. Therefore, EPSPS transcriptional regulation confers glyphosate resistance in this E. indica population. This article is protected by copyright. All rights reserved.

Overexpression of microRNA miR-30a or miR-191 in A549 lung cancer or BEAS-2B normal lung cell lines does not alter phenotype.

Directory of Open Access Journals (Sweden)

Santosh Kumar Patnaik

Full Text Available BACKGROUND: MicroRNAs (miRNAs are small, noncoding RNAs (ribonucleic acids that regulate translation. Several miRNAs have been shown to be altered in whole cancer tissue compared to normal tissue when quantified by microarray. Based on previous such evidence of differential expression, we chose to study the functional significance of miRNAs miR-30a and -191 alterations in human lung cancer. METHODOLOGY/PRINCIPAL FINDINGS: The functional significance of miRNAs miR-30a and -191 was studied by creating stable transfectants of the lung adenocarcinoma cell line A549 and the immortalized bronchial epithelial cell line BEAS-2B with modest overexpression of miR-30a or -191 using a lentiviral system. When compared to the corresponding controls, both cell lines overexpressing miR-30a or -191 do not demonstrate any significant changes in cell cycle distribution, cell proliferation, adherent colony formation, soft agar colony formation, xenograft formation in a subcutaneous SCID mouse model, and drug sensitivity to doxorubicin and cisplatin. There is a modest increase in cell migration in cell lines overexpressing miR-30a compared to their controls. CONCLUSIONS/SIGNIFICANCE: Overexpression of miR-30a or -191 does not lead to an alteration in cell cycle, proliferation, xenograft formation, and chemosensitivity of A549 and BEAS-2B cell lines. Using microarray data from whole tumors to select specific miRNAs for functional study may be a suboptimal strategy.

Solution structure of the major fish allergen parvalbumin Sco j 1 derived from the Pacific mackerel.

Science.gov (United States)

Kumeta, Hiroyuki; Nakayama, Haruka; Ogura, Kenji

2017-12-07

Although fish is an important part of the human diet, it is also a common source of food allergy. The major allergen in fish is parvalbumin, a well-conserved Ca 2+ -binding protein found in the white muscle of many fish species. Here, we studied the solution structure of the parvalbumin Sco j 1, derived from the Pacific mackerel, using nuclear magnetic resonance spectroscopy. We mapped the IgE-binding epitope proposed in a recent study onto the present structure. Interestingly, three of four residues, which were elucidated as key residues of the IgE-binding epitope, were exposed to solvent, whereas one residue faced the inside of the molecule. We expect that this solution structure can be used in future studies attempting to analyze the various IgE-binding modes of these allergens.

Overexpression of suppressor of cytokine signaling 3 in the arcuate nucleus of juvenile Phodopus sungorus alters seasonal body weight changes.

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Ganjam, Goutham K; Benzler, Jonas; Pinkenburg, Olaf; Boucsein, Alisa; Stöhr, Sigrid; Steger, Juliane; Culmsee, Carsten; Barrett, Perry; Tups, Alexander

2013-12-01

The profound seasonal cycle in body weight exhibited by the Djungarian hamster (Phodopus sungorus) is associated with the development of hypothalamic leptin resistance during long day photoperiod (LD, 16:8 h light dark cycle), when body weight is elevated relative to short day photoperiod (SD, 8:16 h light dark cycle). We previously have shown that this seasonal change in physiology is associated with higher levels of mRNA for the potent inhibitor of leptin signaling, suppressor of cytokine signaling-3 (SOCS3), in the arcuate nucleus (ARC) of LD hamsters relative to hamsters in SD. The alteration in SOCS3 gene expression preceded the body weight change suggesting that SOCS3 might be the molecular switch of seasonal body weight changes. To functionally characterize the role of SOCS3 in seasonal body weight regulation, we injected SOCS3 expressing recombinant adeno-associated virus type-2 (rAAV2-SOCS3) constructs into the ARC of leptin sensitive SD hamsters immediately after weaning. Hamsters that received rAAV2 expressing enhanced green fluorescent protein (rAAV2-EGFP) served as controls. ARC-directed SOCS3 overexpression led to a significant increase in body weight over a period of 12 weeks without fully restoring the LD phenotype. This increase was partially due to elevated brown and white adipose tissue mass. Gene expression of pro-opiomelanocortin was increased while thyroid hormone converting enzyme DIO3 mRNA levels were reduced in SD hamsters with SOCS3 overexpression. In conclusion, our data suggest that ARC-directed SOCS3 overexpression partially overcomes the profound seasonal body weight cycle exhibited by the hamster which is associated with altered pro-opiomelanocortin and DIO3 gene expression.

Efferent connections of the parvalbumin-positive (PV1) nucleus in the lateral hypothalamus of rodents.

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Celio, Marco R; Babalian, Alexandre; Ha, Quan Hue; Eichenberger, Simone; Clément, Laurence; Marti, Christiane; Saper, Clifford B

2013-10-01

A solitary cluster of parvalbumin-positive neurons--the PV1 nucleus--has been observed in the lateral hypothalamus of rodents. In the present study, we mapped the efferent connections of the PV1 nucleus using nonspecific antero- and retrograde tracers in rats, and chemoselective, Cre-dependent viral constructs in parvalbumin-Cre mice. In both species, the PV1 nucleus was found to project mainly to the periaqueductal grey matter (PAG), predominantly ipsilaterally. Indirectly in rats and directly in mice, a discrete, longitudinally oriented cylindrical column of terminal fields (PV1-CTF) was identified ventrolateral to the aqueduct on the edge of the PAG. The PV1-CTF is particularly dense in the rostral portion, which is located in the supraoculomotor nucleus (Su3). It is spatially interrupted over a short stretch at the level of the trochlear nucleus and abuts caudally on a second parvalbumin-positive (PV2) nucleus. The rostral and the caudal portions of the PV1-CTF consist of axonal endings, which stem from neurons scattered throughout the PV1 nucleus. Topographically, the longitudinal orientation of the PV1-CTF accords with that of the likewise longitudinally oriented functional modules of the PAG, but overlaps none of them. Minor terminal fields were identified in a crescentic column of the lateral PAG, as well as in the Edinger-Westphal, the lateral habenular, and the laterodorsal tegmental nuclei. So far, no obvious functions have been attributed to this small, circumscribed column ventrolateral to the aqueduct, the prime target of the PV1 nucleus. © 2013 Wiley Periodicals, Inc.

Overexpression of a Panax ginseng tonoplast aquaporin alters salt tolerance, drought tolerance and cold acclimation ability in transgenic Arabidopsis plants.

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Peng, Yanhui; Lin, Wuling; Cai, Weiming; Arora, Rajeev

2007-08-01

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant's response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na(+) compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.

Remodeling of the AB site of rat parvalbumin and oncomodulin into a canonical EF-hand

DEFF Research Database (Denmark)

Cox, J A; Durussel, I; Scott, D J

1999-01-01

-residue canonical loop. To create an optical conformational probe we also expressed the homologs with a F102W replacement. Unexpectedly, in none of the proteins did the mutation reactivate the AB site. The AB-remodeled parvalbumins bind two Ca2+ ions with strong positive cooperativity (nH = 2......Parvalbumin (PV) and the homologous protein oncomodulin (OM) contain three EF-hand motifs, but the first site (AB) cannot bind Ca2+. Here we aimed to recreate the putative ancestral proteins [D19-28E]PV and [D19-28E]OM by replacing the 10-residue-long nonfunctional loop in the AB site by a 12...... conformations. The AB-remodeled oncomodulins also bind two Ca2+ with [Ca2+]0.5 = 43 microM and nH = 1.45. Mg2+ does not affect Ca2+ binding. Again the Ca2+ forms display two-thirds of the alpha-helical content in the wild-type, while their core is still strongly hydrophobic as monitored by Trp and Tyr...

Changes in parvalbumin immunoreactivity with aging in the central auditory system of the rat

Czech Academy of Sciences Publication Activity Database

Ouda, Ladislav; Druga, R.; Syka, Josef

2008-01-01

Roč. 43, č. 8 (2008), s. 782-789 ISSN 0531-5565 R&D Projects: GA ČR GA309/07/1336; GA MZd NR8113; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50390512 Keywords : Calcium binding proteins * Parvalbumin * Aging Subject RIV: FH - Neurology Impact factor: 3.283, year: 2008

DEVELOPMENTAL HYPOTHYROIDISM REDUCES PARVALBUMIN EXPRESSION IN GABAERGIC NEURONS OF CORTEX AND HIPPOCAMPUS: IMMUNOHISTOCHEMICAL FINDINGS AND FUNCTIONAL CORRELATES.

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GABAergic interneurons comprise the bulk of local inhibitory neuronal circuitry in cortex and hippocampus and a subpopulation of these interneurons contain the calcium binding protein, parvalbumin (PV). A previous report indicated that severe hypothyroidism reduced PV immunoreact...

A major allergen in rainbow trout (Oncorhynchus mykiss): complete sequences of parvalbumin by MALDI tandem mass spectrometry.

Science.gov (United States)

Aiello, Donatella; Materazzi, Stefano; Risoluti, Roberta; Thangavel, Hariprasad; Di Donna, Leonardo; Mazzotti, Fabio; Casadonte, Francesca; Siciliano, Carlo; Sindona, Giovanni; Napoli, Anna

2015-08-01

Fish parvalbumin (PRVB) is an abundant and stable protein in fish meat. The variation in cross-reactivity among individuals is well known and explained by a broad repertoire of molecular forms and differences between IgE-binding epitopes in fish species. PVRB has "sequential" epitopes, which retain their IgE-binding capacity and allergenicity also after heating and digestion using proteolytic enzymes. From the allergonomics perspective, PRVB is still a challenging target due to its multiple isoforms present at different degrees of distribution. Little information is available in the databases about PVRBs from Oncorhynchus mykiss. At present, only two validated, incomplete isoforms of this species are included in the protein databases: parvalbumin beta 1 (P86431) and parvalbumin beta 2 (P86432). A simple and rapid protocol has been developed for selective solubilization of PRVB from the muscle of farmed rainbow trout (Oncorhynchus mykiss), followed by calcium depletion, proteolytic digestion, MALDI MS, and MS/MS analysis. With this strategy thermal allergen release was assessed and PRVB1 (P86431), PRVB1.1, PRVB2 (P86432) and PRVB2.1 variants from the rainbow trout were sequenced. The correct ordering of peptide sequences was aided by mapping the overlapping enzymatic digests. The deduced peptide sequences were arranged and the theoretical molecular masses (Mr) of the resulting sequences were calculated. Experimental masses (Mr) of each PRVB variant were measured by linear MALDI-TOF.

Sex matters: females in proestrus show greater diazepam anxiolysis and brain-derived neurotrophin factor- and parvalbumin-positive neurons than males.

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Ravenelle, Rebecca; Berman, Ariel K; La, Jeffrey; Mason, Briana; Asumadu, Evans; Yelleswarapu, Chandra; Donaldson, S Tiffany

2018-04-01

In humans and animal models, sex differences are reported for anxiety-like behavior and response to anxiogenic stimuli. In the current work, we studied anxiety-like behavior and response to the prototypical anti-anxiety drug, diazepam. We used 6th generation outbred lines of adult Long Evans rats with high and low anxiety-like behavior phenotypes to investigate the impact of proestrus on the baseline and diazepam-induced behavior. At three doses of diazepam (0, 0.1, and 1.0 mg/kg, i.p.), we measured anxiogenic responses on the elevated plus maze of adult male and female rats. We assessed parvalbumin and brain-derived neurotrophin protein levels in forebrain and limbic structures implicated in anxiety/stress using immunohistochemistry. At baseline, we saw significant differences between anxiety lines, with high anxiety lines displaying less time on the open arms of the elevated plus maze, and less open arm entries, regardless of sex. During proestrus, high anxiety females showed less anxiety-like behavior at 0.1 mg/kg, while low anxiety females displayed less anxiety-like behavior at 0.1 and 1.0 doses, relative to males. Brain-derived neurotrophin protein was elevated in females in the medial prefrontal cortex and central amygdala, while parvalbumin-immunoreactive cells were greater in males in the medial prefrontal cortex. Parvalbumin-positive cells in high anxiety females were higher in CA2 and dentate gyrus relative to males from the same line. In sum, when tested in proestrus, females showed greater anxiolytic effects of diazepam relative to males, and this correlated with increases in neurotrophin and parvalbumin neuron density in corticolimbic structures. © 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Layer 5 Callosal Parvalbumin-Expressing Neurons: A Distinct Functional Group of GABAergic Neurons.

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Zurita, Hector; Feyen, Paul L C; Apicella, Alfonso Junior

2018-01-01

Previous studies have shown that parvalbumin-expressing neurons (CC-Parv neurons) connect the two hemispheres of motor and sensory areas via the corpus callosum, and are a functional part of the cortical circuit. Here we test the hypothesis that layer 5 CC-Parv neurons possess anatomical and molecular mechanisms which dampen excitability and modulate the gating of interhemispheric inhibition. In order to investigate this hypothesis we use viral tracing to determine the anatomical and electrophysiological properties of layer 5 CC-Parv and parvalbumin-expressing (Parv) neurons of the mouse auditory cortex (AC). Here we show that layer 5 CC-Parv neurons had larger dendritic fields characterized by longer dendrites that branched farther from the soma, whereas layer 5 Parv neurons had smaller dendritic fields characterized by shorter dendrites that branched nearer to the soma. The layer 5 CC-Parv neurons are characterized by delayed action potential (AP) responses to threshold currents, lower firing rates, and lower instantaneous frequencies compared to the layer 5 Parv neurons. Kv1.1 containing K + channels are the main source of the AP repolarization of the layer 5 CC-Parv and have a major role in determining both the spike delayed response, firing rate and instantaneous frequency of these neurons.

Nimodipine accelerates the postnatal development of parvalbumin and S-100β immunoreactivity in the rat brain

NARCIS (Netherlands)

Buwalda, Bauke; Naber, Riet; Nyakas, Csaba; Luiten, Paul G.M.

1994-01-01

The effects of chronic maternal perinatal nimodipine treatment on the immunocytochemical distribution of the Ca2+-binding proteins parvalbumin (PV) and S-100β in neocortex and hippocampus were studied at the age of postnatal day (PD) 5, 7, 10, 14 and 20. The Ca2+ antagonist nimodipine (1000 ppm BAY

Fish allergens at a glance: Variable allergenicity of parvalbumins, the major fish allergens

OpenAIRE

Annette eKuehn; Ines eSwoboda; Karthik eArumugam; Christiane eHilger; François eHentges; François eHentges

2014-01-01

Fish is a common trigger of severe, food-allergic reactions. Only a limited number of proteins induce specific IgE-mediated immune reactions. The major fish allergens are the parvalbumins. They are members of the calcium-binding EF-hand protein family characterized by a conserved protein structure. They represent highly cross-reactive allergens for patients with specific IgE to conserved epitopes. These patients might experience clinical reactions with various fish species. On the other hand,...

Fish Allergens at a Glance: Variable Allergenicity of Parvalbumins, the Major Fish Allergens

OpenAIRE

Kuehn, Annette; Swoboda, Ines; Arumugam, Karthik; Hilger, Christiane; Hentges, François

2014-01-01

Fish is a common trigger of severe, food-allergic reactions. Only a limited number of proteins induce specific IgE-mediated immune reactions. The major fish allergens are the parvalbumins. They are members of the calcium-binding EF-hand protein family characterized by a conserved protein structure. They represent highly cross-reactive allergens for patients with specific IgE to conserved epitopes. These patients might experience clinical reactions with various fish species. On the other hand,...

Identification of parvalbumin and two new thermolabile major allergens of Thunnus tonggol using a proteomics approach.

Science.gov (United States)

Rosmilah, Misnan; Shahnaz, Murad; Meinir, Jones; Masita, Arip; Noormalin, Abdullah; Jamaluddin, Mohamed

2013-01-01

The longtail tuna (Thunnus tonggol) is widely consumed in Asia. Parvalbumin, the main major allergen of fish, has been well identified in multiple fish species, yet little is known about the allergenic proteins in T. tonggol. Thus, the aim of this study was to characterize the major allergens of T. tonggol using a proteomics approach. Raw and boiled extracts of the fish were prepared. Fish proteins were separated by means of SDS-PAGE and two-dimensional (2-DE) electrophoresis. 1-DE immunoblotting of raw extract was performed with sera from fish-allergic patients. Ten sera were further analysed by 2-DE immunoblotting. Selected major allergenic protein spots were excised, trypsin digested and analysed by means of mass spectrometry. SDS-PAGE of raw extract revealed 26 protein fractions, while boiled extract demonstrated fewer bands. The 2-DE gel profile of the raw extract further fractionated the protein bands to more than 100 distinct protein spots. 1-DE immunoblotting of raw extract exhibited two thermolabile protein fractions of 42 and 51 kDa as the major allergens, while the boiled extract only revealed a single IgE-binding band at 151 kDa. 2-DE immunoblotting of raw extract further detected numerous major IgE-reactive spots of 11-13, 42 and 51 kDa. Mass spectrometry analysis of the peptides generated from the 12, 42 and 51 kDa digested spots indicated that these spots were parvalbumin, creatine kinase and enolase, respectively. In addition to parvalbumin, two new thermolabile allergens were identified as major allergenic proteins of T. tonggol. This study proved that both thermostable and thermolabile proteins are important in local tuna allergy and should be included in diagnostic strategies.

Calretinin and parvalbumin immunoreactive interneurons in the retrosplenial cortex of the rat brain: Qualitative and quantitative analyses

Czech Academy of Sciences Publication Activity Database

Salaj, M.; Druga, Rastislav; Cerman, J.; Kubová, Hana; Barinka, F.

2015-01-01

Roč. 1627, Nov 19 (2015), s. 201-215 ISSN 0006-8993 R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : retrosplenial cortex * calretinin * parvalbumin * interneurons * calcium-binding proteins * perirhinal cortex Subject RIV: FH - Neurology Impact factor: 2.561, year: 2015

Alterations in Adiposity and Glucose Homeostasis in Adult Gasp-1 Overexpressing Mice

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Luce Périè

2017-12-01

Full Text Available Background/Aims: Myostatin is known as a powerful negative regulator of muscle growth playing a key role in skeletal muscle homeostasis. Recent studies revealed that myostatin-deficient mice lead to an increase of insulin sensitivity, a decrease of adiposity and a resistance to obesity, showing that myostatin can also impact on metabolism. Thus, myostatin appeared as a potential therapeutic target to treat insulin resistance. Methods: We generated transgenic mice overexpressing Gasp-1, a myostatin inhibitor. Results: Surprisingly, we found that these mice gained weight with age due to an increase in fat mass associated with ectopic fat accumulation. In addition, these mice developed an adipocyte hypertrophy, hyperglycemia, hyperinsulinemia, muscle and hepatic insulin resistance. Understanding the molecular networks controlling this insulin resistance responsiveness in overexpressing Gasp-1 mice is essential. Molecular analyses revealed a deregulation of adipokines and muscle cytokines expression, but also an increase in plasma myostatin levels. The increase in myostatin bioactivity by a positive feedback mechanism in the Tg(Gasp-1 transgenic mice could lead to this combination of phenotypes. Conclusion: Altogether, these data suggested that overexpressing Gasp-1 mice develop most of the symptoms associated with metabolic syndrome and could be a relevant model for the study of obesity or type 2 diabetes.

Transgenic overexpression of adenosine kinase in brain leads to multiple learning impairments and altered sensitivity to psychomimetic drugs.

Science.gov (United States)

Yee, Benjamin K; Singer, Philipp; Chen, Jiang-Fan; Feldon, Joram; Boison, Detlev

2007-12-01

The neuromodulator adenosine fulfills a unique role in the brain affecting glutamatergic neurotransmission and dopaminergic signaling via activation of adenosine A1 and A2A receptors, respectively. The adenosine system is thus ideally positioned to integrate glutamatergic and dopaminergic neurotransmission, which in turn could affect behavior and cognition. In the adult brain, adenosine levels are largely regulated by its key metabolic enzyme adenosine kinase (ADK), which may assume the role of an 'upstream regulator' of these two neurotransmitter pathways. To test this hypothesis, transgenic mice with an overexpression of ADK in brain (Adk-tg), and therefore reduced brain adenosine levels, were evaluated in a panel of behavioral and psychopharmacological assays to assess possible glutamatergic and dopaminergic dysfunction. In comparison to non-transgenic control mice, Adk-tg mice are characterized by severe learning deficits in the Morris water maze task and in Pavlovian conditioning. The Adk-tg mice also exhibited reduced locomotor reaction to systemic amphetamine, whereas their reaction to the non-competitive N-methyl-d-aspartate receptor antagonist MK-801 was enhanced. Our results confirmed that ADK overexpression could lead to functional concomitant alterations in dopaminergic and glutamatergic functions, which is in keeping with the hypothesized role of ADK in the balance and integration between glutamatergic and dopaminergic neurotransmission. The present findings are of relevance to current pathophysiological hypotheses of schizophrenia and its pharmacotherapy.

Bone turnover is altered in transgenic rats overexpressing the P2Y2 purinergic receptor

DEFF Research Database (Denmark)

Ellegaard, Maria; Agca, Cansu; Petersen, Solveig

2017-01-01

overexpression on bone status and bone cell function using a transgenic rat. Three-month-old female transgenic Sprague Dawley rats overexpressing P2Y2R (P2Y2R-Tg) showed higher bone strength of the femoral neck. Histomorphometry showed increase in resorptive surfaces and reduction in mineralizing surfaces. Both...

Singing modulates parvalbumin interneurons throughout songbird forebrain vocal control circuitry

Science.gov (United States)

Zengin-Toktas, Yildiz

2017-01-01

Across species, the performance of vocal signals can be modulated by the social environment. Zebra finches, for example, adjust their song performance when singing to females (‘female-directed’ or FD song) compared to when singing in isolation (‘undirected’ or UD song). These changes are salient, as females prefer the FD song over the UD song. Despite the importance of these performance changes, the neural mechanisms underlying this social modulation remain poorly understood. Previous work in finches has established that expression of the immediate early gene EGR1 is increased during singing and modulated by social context within the vocal control circuitry. Here, we examined whether particular neural subpopulations within those vocal control regions exhibit similar modulations of EGR1 expression. We compared EGR1 expression in neurons expressing parvalbumin (PV), a calcium buffer that modulates network plasticity and homeostasis, among males that performed FD song, males that produced UD song, or males that did not sing. We found that, overall, singing but not social context significantly affected EGR1 expression in PV neurons throughout the vocal control nuclei. We observed differences in EGR1 expression between two classes of PV interneurons in the basal ganglia nucleus Area X. Additionally, we found that singing altered the amount of PV expression in neurons in HVC and Area X and that distinct PV interneuron types in Area X exhibited different patterns of modulation by singing. These data indicate that throughout the vocal control circuitry the singing-related regulation of EGR1 expression in PV neurons may be less influenced by social context than in other neuron types and raise the possibility of cell-type specific differences in plasticity and calcium buffering. PMID:28235074

15N NMR relaxation studies of calcium-loaded parvalbumin show tight dynamics compared to those of other EF-hand proteins

DEFF Research Database (Denmark)

Baldellon, C; Alattia, J R; Strub, M P

1998-01-01

Dynamics of the rat alpha-parvalbumin calcium-loaded form have been determined by measurement of 15N nuclear relaxation using proton-detected heteronuclear NMR spectroscopy. The relaxation data were analyzed using spectral density functions and the Lipari-Szabo formalism. The major dynamic features...... for the rat alpha-parvalbumin calcium-loaded form are (1) the extreme rigidity of the helix-loop-helix EF-hand motifs and the linker segment connecting them, (2) the N and C termini of the protein being restricted in their mobility, (3) a conformational exchange occurring at the kink of helix D, and (4...... properties which are conserved in the EF-hand domains from different members of this superfamily: (1) a tendency toward higher mobility of NH vectors at relative position 2 in the Ca2+-binding loop, (2) a restricted mobility for the other residues in the binding loop, and (3) an overall rigidity...

Constitutive overexpression of muscarinic receptors leads to vagal hyperreactivity.

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Angelo Livolsi

Full Text Available BACKGROUND: Alterations in muscarinic receptor expression and acetylcholinesterase (AchE activity have been observed in tissues from Sudden Infant Death Syndrome (SIDS. Vagal overactivity has been proposed as a possible cause of SIDS as well as of vasovagal syncopes. The aim of the present study was to seek whether muscarinic receptor overexpression may be the underlying mechanism of vagal hyperreactivity. Rabbits with marked vagal pauses following injection of phenylephrine were selected and crossed to obtain a vagal hyperreactive strain. The density of cardiac muscarinic receptors and acetylcholinesterase (AchE gene expression were assessed. Blood markers of the observed cardiac abnormalities were also sought. METHODOLOGY/PRINCIPAL FINDINGS: Cardiac muscarinic M(2 and M(3 receptors were overexpressed in hyperreactive rabbits compared to control animals (2.3-fold and 2.5-fold, respectively and the severity of the phenylephrine-induced bradycardia was correlated with their densities. A similar overexpression of M(2 receptors was observed in peripheral mononuclear white blood cells, suggesting that cardiac M(2 receptor expression can be inferred with high confidence from measurements in blood cells. Sequencing of the coding fragment of the M(2 receptor gene revealed a single nucleotide mutation in 83% of hyperreactive animals, possibly contributing for the transcript overexpression. Significant increases in AchE expression and activity were also assessed (AchE mRNA amplification ratio of 3.6 versus normal rabbits. This phenomenon might represent a compensatory consequence of muscarinic receptors overexpression. Alterations in M(2 receptor and AchE expression occurred between the 5th and the 7th week of age, a critical period also characterized by a higher mortality rate of hyperreactive rabbits (52% in H rabbits versus 13% in normal rabbits and preceeded the appearance of functional disorders. CONCLUSIONS/SIGNIFICANCE: The results suggest that

Profilin-1 overexpression in MDA-MB-231 breast cancer cells is associated with alterations in proteomics biomarkers of cell proliferation, survival, and motility as revealed by global proteomics analyses.

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Coumans, Joëlle V F; Gau, David; Poljak, Anne; Wasinger, Valerie; Roy, Partha; Moens, Pierre D J

2014-12-01

Despite early screening programs and new therapeutic strategies, metastatic breast cancer is still the leading cause of cancer death in women in industrialized countries and regions. There is a need for novel biomarkers of susceptibility, progression, and therapeutic response. Global analyses or systems science approaches with omics technologies offer concrete ways forward in biomarker discovery for breast cancer. Previous studies have shown that expression of profilin-1 (PFN1), a ubiquitously expressed actin-binding protein, is downregulated in invasive and metastatic breast cancer. It has also been reported that PFN1 overexpression can suppress tumorigenic ability and motility/invasiveness of breast cancer cells. To obtain insights into the underlying molecular mechanisms of how elevating PFN1 level induces these phenotypic changes in breast cancer cells, we investigated the alteration in global protein expression profiles of breast cancer cells upon stable overexpression of PFN1 by a combination of three different proteome analysis methods (2-DE, iTRAQ, label-free). Using MDA-MB-231 as a model breast cancer cell line, we provide evidence that PFN1 overexpression is associated with alterations in the expression of proteins that have been functionally linked to cell proliferation (FKPB1A, HDGF, MIF, PRDX1, TXNRD1, LGALS1, STMN1, LASP1, S100A11, S100A6), survival (HSPE1, HSPB1, HSPD1, HSPA5 and PPIA, YWHAZ, CFL1, NME1) and motility (CFL1, CORO1B, PFN2, PLS3, FLNA, FLNB, NME2, ARHGDIB). In view of the pleotropic effects of PFN1 overexpression in breast cancer cells as suggested by these new findings, we propose that PFN1-induced phenotypic changes in cancer cells involve multiple mechanisms. Our data reported here might also offer innovative strategies for identification and validation of novel therapeutic targets and companion diagnostics for persons with, or susceptibility to, breast cancer.

General applicability of synthetic gene-overexpression for cell-type ratio control via reprogramming.

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Ishimatsu, Kana; Hata, Takashi; Mochizuki, Atsushi; Sekine, Ryoji; Yamamura, Masayuki; Kiga, Daisuke

2014-09-19

Control of the cell-type ratio in multistable systems requires wide-range control of the initial states of cells. Here, using a synthetic circuit in E. coli, we describe the use of a simple gene-overexpression system combined with a bistable toggle switch, for the purposes of enabling the wide-range control of cellular states and thus generating arbitrary cell-type ratios. Theoretically, overexpression induction temporarily alters the bistable system to a monostable system, in which the location of the single steady state of cells can be manipulated over a wide range by regulating the overexpression levels. This induced cellular state becomes the initial state of the basal bistable system upon overexpression cessation, which restores the original bistable system. We experimentally demonstrated that the overexpression induced a monomodal cell distribution, and subsequent overexpression withdrawal generated a bimodal distribution. Furthermore, as designed theoretically, regulating the overexpression levels by adjusting the concentrations of small molecules generated arbitrary cell-type ratios.

GABA Neuron Alterations, Cortical Circuit Dysfunction and Cognitive Deficits in Schizophrenia

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Guillermo Gonzalez-Burgos

2011-01-01

Full Text Available Schizophrenia is a brain disorder associated with cognitive deficits that severely affect the patients' capacity for daily functioning. Whereas our understanding of its pathophysiology is limited, postmortem studies suggest that schizophrenia is associated with deficits of GABA-mediated synaptic transmission. A major role of GABA-mediated transmission may be producing synchronized network oscillations which are currently hypothesized to be essential for normal cognitive function. Therefore, cognitive deficits in schizophrenia may result from a GABA synapse dysfunction that disturbs neural synchrony. Here, we highlight recent studies further suggesting alterations of GABA transmission and network oscillations in schizophrenia. We also review current models for the mechanisms of GABA-mediated synchronization of neural activity, focusing on parvalbumin-positive GABA neurons, which are altered in schizophrenia and whose function has been strongly linked to the production of neural synchrony. Alterations of GABA signaling that impair gamma oscillations and, as a result, cognitive function suggest paths for novel therapeutic interventions.

GABA neuron alterations, cortical circuit dysfunction and cognitive deficits in schizophrenia.

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Gonzalez-Burgos, Guillermo; Fish, Kenneth N; Lewis, David A

2011-01-01

Schizophrenia is a brain disorder associated with cognitive deficits that severely affect the patients' capacity for daily functioning. Whereas our understanding of its pathophysiology is limited, postmortem studies suggest that schizophrenia is associated with deficits of GABA-mediated synaptic transmission. A major role of GABA-mediated transmission may be producing synchronized network oscillations which are currently hypothesized to be essential for normal cognitive function. Therefore, cognitive deficits in schizophrenia may result from a GABA synapse dysfunction that disturbs neural synchrony. Here, we highlight recent studies further suggesting alterations of GABA transmission and network oscillations in schizophrenia. We also review current models for the mechanisms of GABA-mediated synchronization of neural activity, focusing on parvalbumin-positive GABA neurons, which are altered in schizophrenia and whose function has been strongly linked to the production of neural synchrony. Alterations of GABA signaling that impair gamma oscillations and, as a result, cognitive function suggest paths for novel therapeutic interventions.

Overexpression of cypin alters dendrite morphology, single neuron activity, and network properties via distinct mechanisms

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Rodríguez, Ana R.; O'Neill, Kate M.; Swiatkowski, Przemyslaw; Patel, Mihir V.; Firestein, Bonnie L.

2018-02-01

Objective. This study investigates the effect that overexpression of cytosolic PSD-95 interactor (cypin), a regulator of synaptic PSD-95 protein localization and a core regulator of dendrite branching, exerts on the electrical activity of rat hippocampal neurons and networks. Approach. We cultured rat hippocampal neurons and used lipid-mediated transfection and lentiviral gene transfer to achieve high levels of cypin or cypin mutant (cypinΔPDZ PSD-95 non-binding) expression cellularly and network-wide, respectively. Main results. Our analysis revealed that although overexpression of cypin and cypinΔPDZ increase dendrite numbers and decrease spine density, cypin and cypinΔPDZ distinctly regulate neuronal activity. At the single cell level, cypin promotes decreases in bursting activity while cypinΔPDZ reduces sEPSC frequency and further decreases bursting compared to cypin. At the network level, by using the Fano factor as a measure of spike count variability, cypin overexpression results in an increase in variability of spike count, and this effect is abolished when cypin cannot bind PSD-95. This variability is also dependent on baseline activity levels and on mean spike rate over time. Finally, our spike sorting data show that overexpression of cypin results in a more complex distribution of spike waveforms and that binding to PSD-95 is essential for this complexity. Significance. Our data suggest that dendrite morphology does not play a major role in cypin action on electrical activity.

Overexpression of BMP3 in the developing skeleton alters endochondral bone formation resulting in spontaneous rib fractures.

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Gamer, Laura W; Cox, Karen; Carlo, Joelle M; Rosen, Vicki

2009-09-01

Bone morphogenetic protein-3 (BMP) has been identified as a negative regulator in the skeleton as mice lacking BMP3 have increased bone mass. To further understand how BMP3 mediates bone formation, we created transgenic mice overexpressing BMP3 using the type I collagen promoter. BMP3 transgenic mice displayed spontaneous rib fractures that were first detected at E17.0. The fractures were due to defects in differentiation of the periosteum and late hypertrophic chondrocytes resulting in thinner cortical bone with decreased mineralization. As BMP3 modulates BMP and activin signaling through ActRIIB, we examined the ribs of ActRIIB receptor knockout mice and found they had defects in late chondrogenesis and mineralization similar to BMP3 transgenic mice. These data suggest that BMP3 exerts its effects in the skeleton by altering signaling through ActRIIB in chondrocytes and the periosteum, and this results in defects in bone collar formation and late hypertrophic chondrocyte maturation leading to decreased mineralization and less bone. 2009 Wiley-Liss, Inc.

Oncoprotein MDM2 Overexpression is Associated with Poor Prognosis in Distinct Non-Hodgkin's Lymphoma Entities

DEFF Research Database (Denmark)

Møller, Michael Boe; Nielsen, O; Pedersen, Niels Tinggaard

1999-01-01

MDM2 is an oncoprotein involved in the regulation of p53. MDM2 exerts its tumorigenic potential through p53-dependent and -independent mechanisms. It is frequently overexpressed in various malignancies. Little is known about the prognostic value of MDM2 expression in non-Hodgkin's lymphomas (NHL...... overexpression was present in 42 (22%) of 188 cases. The frequency was highest in aggressive/very aggressive NHL (P lymphomas, MDM2 overexpression was associated with higher-grade disease (P = .008). MDM2 overexpression was not related to a phenotype indicating...... altered p53. In univariate analysis MDM2 overexpression associated with short survival in follicle center lymphomas (P = .0256), extranodal marginal zone lymphomas (P lymphomas (P = .0047). The relation to poor prognosis was maintained in a Cox regression analysis including known...

Overexpression of the essential Sis1 chaperone reduces TDP-43 effects on toxicity and proteolysis

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Park, Sei-Kyoung; Hong, Joo Y.; Arslan, Fatih; Tietsort, Alex; Tank, Elizabeth M. H.; Li, Xingli

2017-01-01

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by selective loss of motor neurons with inclusions frequently containing the RNA/DNA binding protein TDP-43. Using a yeast model of ALS exhibiting TDP-43 dependent toxicity, we now show that TDP-43 overexpression dramatically alters cell shape and reduces ubiquitin dependent proteolysis of a reporter construct. Furthermore, we show that an excess of the Hsp40 chaperone, Sis1, reduced TDP-43’s effect on toxicity, cell shape and proteolysis. The strength of these effects was influenced by the presence of the endogenous yeast prion, [PIN+]. Although overexpression of Sis1 altered the TDP-43 aggregation pattern, we did not detect physical association of Sis1 with TDP-43, suggesting the possibility of indirect effects on TDP-43 aggregation. Furthermore, overexpression of the mammalian Sis1 homologue, DNAJB1, relieves TDP-43 mediated toxicity in primary rodent cortical neurons, suggesting that Sis1 and its homologues may have neuroprotective effects in ALS. PMID:28531192

ATG5 overexpression is neuroprotective and attenuates cytoskeletal and vesicle-trafficking alterations in axotomized motoneurons.

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Leiva-Rodríguez, Tatiana; Romeo-Guitart, David; Marmolejo-Martínez-Artesero, Sara; Herrando-Grabulosa, Mireia; Bosch, Assumpció; Forés, Joaquim; Casas, Caty

2018-05-24

Injured neurons should engage endogenous mechanisms of self-protection to limit neurodegeneration. Enhancing efficacy of these mechanisms or correcting dysfunctional pathways may be a successful strategy for inducing neuroprotection. Spinal motoneurons retrogradely degenerate after proximal axotomy due to mechanical detachment (avulsion) of the nerve roots, and this limits recovery of nervous system function in patients after this type of trauma. In a previously reported proteomic analysis, we demonstrated that autophagy is a key endogenous mechanism that may allow motoneuron survival and regeneration after distal axotomy and suture of the nerve. Herein, we show that autophagy flux is dysfunctional or blocked in degenerated motoneurons after root avulsion. We also found that there were abnormalities in anterograde/retrograde motor proteins, key secretory pathway factors, and lysosome function. Further, LAMP1 protein was missorted and underglycosylated as well as the proton pump v-ATPase. In vitro modeling revealed how sequential disruptions in these systems likely lead to neurodegeneration. In vivo, we observed that cytoskeletal alterations, induced by a single injection of nocodazole, were sufficient to promote neurodegeneration of avulsed motoneurons. Besides, only pre-treatment with rapamycin, but not post-treatment, neuroprotected after nerve root avulsion. In agreement, overexpressing ATG5 in injured motoneurons led to neuroprotection and attenuation of cytoskeletal and trafficking-related abnormalities. These discoveries serve as proof of concept for autophagy-target therapy to halting the progression of neurodegenerative processes.

Overexpression of BiP in tobacco alleviates endoplasmic reticulum stress

NARCIS (Netherlands)

Leborgne-Castel, N.; Jelitto- Dooren, E.P.W.M. van; Crofts, A.J.; Denecke, J.

1999-01-01

To study the role of the lumenal binding protein (BiP) in the transport and secretion of proteins, we have produced plants with altered BiP levels. Transgenic plants overexpressing BiP showed dramatically increased BiP mRNA levels but only a modest increase in BiP protein levels. The presence of

Targeted overexpression of amelotin disrupts the microstructure of dental enamel.

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Lacruz, Rodrigo S; Nakayama, Yohei; Holcroft, James; Nguyen, Van; Somogyi-Ganss, Eszter; Snead, Malcolm L; White, Shane N; Paine, Michael L; Ganss, Bernhard

2012-01-01

We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.

Targeted overexpression of amelotin disrupts the microstructure of dental enamel.

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Rodrigo S Lacruz

Full Text Available We have previously identified amelotin (AMTN as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL and ameloblastin (AMBN was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.

ERAP1 overexpression in HPV-induced malignancies: A possible novel immune evasion mechanism.

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Steinbach, Alina; Winter, Jan; Reuschenbach, Miriam; Blatnik, Renata; Klevenz, Alexandra; Bertrand, Miriam; Hoppe, Stephanie; von Knebel Doeberitz, Magnus; Grabowska, Agnieszka K; Riemer, Angelika B

2017-01-01

Immune evasion of tumors poses a major challenge for immunotherapy. For human papillomavirus (HPV)-induced malignancies, multiple immune evasion mechanisms have been described, including altered expression of antigen processing machinery (APM) components. These changes can directly influence epitope presentation and thus T-cell responses against tumor cells. To date, the APM had not been studied systematically in a large array of HPV + tumor samples. Therefore in this study, systematic expression analysis of the APM was performed on the mRNA and protein level in a comprehensive collection of HPV16 + cell lines. Subsequently, HPV + cervical tissue samples were examined by immunohistochemistry. ERAP1 (endoplasmic reticulum aminopeptidase 1) was the only APM component consistently altered - namely overexpressed - in HPV16 + tumor cell lines. ERAP1 was also found to be overexpressed in cervical intraepithelial neoplasia and cervical cancer samples; expression levels were increasing with disease stage. On the functional level, the influence of ERAP1 expression levels on HPV16 E7-derived epitope presentation was investigated by mass spectrometry and in cytotoxicity assays with HPV16-specific T-cell lines. ERAP1 overexpression did not cause a complete destruction of any of the HPV epitopes analyzed, however, an influence of ERAP1 overexpression on the presentation levels of certain HPV epitopes could be demonstrated by HPV16-specific CD8 + T-cells. These showed enhanced killing toward HPV16 + CaSki cells whose ERAP1 expression had been attenuated to normal levels. ERAP1 overexpression may thus represent a novel immune evasion mechanism in HPV-induced malignancies, in cases when presentation of clinically relevant epitopes is reduced by overactivity of this peptidase.

Niemann-pick type C1 (NPC1) overexpression alters cellular cholesterol homeostasis.

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Millard, E E; Srivastava, K; Traub, L M; Schaffer, J E; Ory, D S

2000-12-08

The Niemann-Pick type C1 (NPC1) protein is a key participant in intracellular trafficking of low density lipoprotein cholesterol, but its role in regulation of sterol homeostasis is not well understood. To characterize further the function of NPC1, we generated stable Chinese hamster ovary (CHO) cell lines overexpressing the human NPC1 protein (CHO/NPC1). NPC1 overexpression increases the rate of trafficking of low density lipoprotein cholesterol to the endoplasmic reticulum and the rate of delivery of endosomal cholesterol to the plasma membrane (PM). CHO/NPC1 cells exhibit a 1.5-fold increase in total cellular cholesterol and up to a 2.9-fold increase in PM cholesterol. This increase in PM cholesterol is closely paralleled by a 3-fold increase in de novo cholesterol synthesis. Inhibition of cholesterol synthesis results in marked redistribution of PM cholesterol to intracellular sites, suggesting an unsuspected role for NPC1 in internalization of PM cholesterol. Despite elevated total cellular cholesterol, CHO/NPC1 cells exhibit increased cholesterol synthesis, which may be attributable to both resistance to oxysterol suppression of sterol-regulated gene expression and to reduced endoplasmic reticulum cholesterol levels under basal conditions. Taken together, these studies provide important new insights into the role of NPC1 in the determination of the levels and distribution of cellular cholesterol.

Solid-state NMR spin-echo investigation of the metalloproteins parvalbumin, concanavalin A, and pea and lentil lectins, substituted with cadmium-113

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Marchetti, Paul S.; Bhattacharyya, Lokesh; Ellis, Paul D.; Brewer, C. Fred

Solid-state 113Cd NMR spectroscopy of static powder samples of 113Cd-substituted metalloproteins, parvalbumin, concanavalin A, and pea and lentil lectins, was carried out. Cross polarization followed by application of a train of uniformly spaced π pulses was employed to investigate the origin of residual cadmium NMR linewidths observed previously in these proteins. Fourier transformation of the resulting spin-echo train yielded spectra consisting of uniformly spaced lines having linewidths of the order of 1-2 ppm. The observed linewidths were not influenced by temperature as low as -50°C or by extent of protein hydration. Since the echo-train pulse sequence is able to eliminate inhomogeneous but not homogeneous contributions to the linewidths, there is a predominant inhomogeneous contribution to cadmium linewidths in the protein CP/MAS spectra. However, significant changes in spectral intensities were observed with change in temperature and extent of protein hydration. These intensity changes are attributed for parvalbumin and concanavalin A to changes in cross-polarization efficiency with temperature and hydration. For pea and lentil lectins, this effect is attributed to the elimination of static disorder at the pea and lentil S2 metal-ion sites due to sugar binding.

PEP3 overexpression shortens lag phase but does not alter growth rate in Saccharomyces cerevisiae exposed to acetic acid stress

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Ding, Jun; Holzwarth, Garrett; Bradford, C. Samuel; Cooley, Ben; Yoshinaga, Allen S.; Patton-Vogt, Jana; Abeliovich, Hagai; Penner, Michael H.; Bakalinsky, Alan T.

2017-01-01

In fungi, two recognized mechanisms contribute to pH homeostasis: the plasma membrane proton-pumping ATPase that exports excess protons and the vacuolar proton-pumping ATPase (V-ATPase) that mediates vacuolar proton uptake. Here, we report that overexpression of PEP3 which encodes a component of the HOPS and CORVET complexes involved in vacuolar biogenesis, shortened lag phase in Saccharomyces cerevisiae exposed to acetic acid stress. By confocal microscopy, PEP3-overexpressing cells stained with the vacuolar membrane-specific dye, FM4-64 had more fragmented vacuoles than the wild-type control. The stained overexpression mutant was also found to exhibit about 3.6-fold more FM4-64 fluorescence than the wild-type control as determined by flow cytometry. While the vacuolar pH of the wild-type strain grown in the presence of 80 mM acetic acid was significantly higher than in the absence of added acid, no significant difference was observed in vacuolar pH of the overexpression strain grown either in the presence or absence of 80 mM acetic acid. Based on an indirect growth assay, the PEP3-overexpression strain exhibited higher V-ATPase activity. We hypothesize that PEP3 overexpression provides protection from acid stress by increasing vacuolar surface area and V-ATPase activity and, hence, proton-sequestering capacity. PMID:26051671

Overexpression of AtGRDP2, a novel glycine-rich domain protein, accelerates plant growth and improves stress tolerance

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Maria Azucena Ortega-Amaro

2015-01-01

Full Text Available Proteins with glycine-rich signatures have been reported in a wide variety of organisms including plants, mammalians, fungi, and bacteria. Plant glycine-rich protein genes exhibit developmental and tissue-specific expression patterns. Herein, we present the characterization of the AtGRDP2 gene using Arabidopsis null and knockdown mutants and, Arabidopsis and lettuce over-expression lines. AtGRDP2 encodes a short glycine-rich domain protein, containing a DUF1399 domain and a putative RNA recognition motif. AtGRDP2 transcript is mainly expressed in Arabidopsis floral organs, and its deregulation in Arabidopsis Atgrdp2 mutants and 35S::AtGRDP2 over-expression lines produces alterations in development. The 35S::AtGRDP2 over-expression lines grow faster than the WT, while the Atgrdp2 mutants have a delay in growth and development. The over-expression lines accumulate higher levels of indole-3-acetic acid and, have alterations in the expression pattern of ARF6, ARF8 and miR167 regulators of floral development and auxin signaling. Under salt stress conditions, 35S::AtGRDP2 over-expression lines displayed higher tolerance and increased expression of stress marker genes. Likewise, transgenic lettuce plants over-expressing the AtGRDP2 gene manifest increased growth rate and early flowering time. Our data reveal an important role for AtGRDP2 in Arabidopsis development and stress response, and suggest a connection between AtGRDP2 and auxin signaling.

Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy

OpenAIRE

Freidl, Raphaela; Gstoettner, Antonia; Baranyi, Ulrike; Swoboda, Ines; Stolz, Frank; Focke-Tejkl, Margarete; Wekerle, Thomas; van Ree, Ronald; Valenta, Rudolf; Linhart, Birgit

2016-01-01

Background Fish is a frequent elicitor of severe IgE-mediated allergic reactions. Beside avoidance, there is currently no allergen-specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium-binding sites have been developed. Objectives This study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunizat...

Transcriptional dysregulation of γ-aminobutyric acid transporter in parvalbumin-containing inhibitory neurons in the prefrontal cortex in schizophrenia.

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Bitanihirwe, Byron K Y; Woo, Tsung-Ung W

2014-12-30

Parvalbumin (PV)-containing neurons are functionally compromised in schizophrenia. Using double in situ hybridization in postmortem human prefrontal cortex, we found that the messenger RNA (mRNA) for the γ-aminobutyric acid (GABA) transporter GAT-1 was undetectable in 22-41% of PV neurons in layers 3-4 in schizophrenia. In the remaining PV neurons with detectable GAT-1 mRNA, transcript expression was decreased by 26% in layer 3. Hence, the dysfunction of PV neurons involves the molecular dysregulation of presynaptic GABA reuptake. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

APRIL is overexpressed in cancer: link with tumor progression

International Nuclear Information System (INIS)

Moreaux, Jérôme; Veyrune, Jean-Luc; De Vos, John; Klein, Bernard

2009-01-01

BAFF and APRIL share two receptors – TACI and BCMA – and BAFF binds to a third receptor, BAFF-R. Increased expression of BAFF and APRIL is noted in hematological malignancies. BAFF and APRIL are essential for the survival of normal and malignant B lymphocytes, and altered expression of BAFF or APRIL or of their receptors (BCMA, TACI, or BAFF-R) have been reported in various B-cell malignancies including B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's lymphoma, multiple myeloma, and Waldenstrom's macroglobulinemia. We compared the expression of BAFF, APRIL, TACI and BAFF-R gene expression in 40 human tumor types – brain, epithelial, lymphoid, germ cells – to that of their normal tissue counterparts using publicly available gene expression data, including the Oncomine Cancer Microarray database. We found significant overexpression of TACI in multiple myeloma and thyroid carcinoma and an association between TACI expression and prognosis in lymphoma. Furthermore, BAFF and APRIL are overexpressed in many cancers and we show that APRIL expression is associated with tumor progression. We also found overexpression of at least one proteoglycan with heparan sulfate chains (HS), which are coreceptors for APRIL and TACI, in tumors where APRIL is either overexpressed or is a prognostic factor. APRIL could induce survival or proliferation directly through HS proteoglycans. Taken together, these data suggest that APRIL is a potential prognostic factor for a large array of malignancies

Purification and characterization of parvalbumins from silver carp (Hypophthalmichthy molitrix).

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Liu, Guang-Ming; Wang, Ning; Cai, Qiu-Feng; Li, Teng; Sun, Le-Chang; Su, Wen-Jin; Cao, Min-Jie

2010-04-30

As the largest producer and consumer of freshwater fish in the world, many people suffer from allergy for consuming freshwater fish in China. However, the allergen profiles of freshwater fish are rarely known. Parvalbumins (PVs) from the white muscle of silver carp (Hypophthalmichthy molitrix) were purified by ammonium sulfate fractionation and column chromatography including DEAE-Sepharose and Superdex 75. Three PV isoforms-PV-I, PV-II, and PV-III-were obtained and their molecular masses as estimated by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 12, 11, and 14 kDa, respectively. All the PVs could be detected by anti-frog PV monoclonal antibody. PV-I and PV-II were quite possibly glycoproteins, while PV-III was not glycosylated, as analyzed by periodic acid-Schiff (PAS) staining. Thermal stability revealed that PV-I and PV-II easily formed polymers, while these proteins were stable in a pH range of 4.0-10.0. A PV gene encoding 110 amino acid residues was cloned and it revealed high identity with PVs from other species of fish. Three isotypes of PV were purified to homogeneity and one distinct PV gene was cloned in silver carp white muscle.

Quantitative nature of overexpression experiments

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Moriya, Hisao

2015-01-01

Overexpression experiments are sometimes considered as qualitative experiments designed to identify novel proteins and study their function. However, in order to draw conclusions regarding protein overexpression through association analyses using large-scale biological data sets, we need to recognize the quantitative nature of overexpression experiments. Here I discuss the quantitative features of two different types of overexpression experiment: absolute and relative. I also introduce the four primary mechanisms involved in growth defects caused by protein overexpression: resource overload, stoichiometric imbalance, promiscuous interactions, and pathway modulation associated with the degree of overexpression. PMID:26543202

Overexpression of Catalase Enhances Benzo(a)pyrene Detoxification in Endothelial Microsomes.

Science.gov (United States)

Yang, Fang; Yang, Hong; Ramesh, Aramandla; Goodwin, J Shawn; Okoro, Emmanuel U; Guo, ZhongMao

2016-01-01

We previously reported that overexpression of catalase upregulated xenobiotic- metabolizing enzyme (XME) expression and diminished benzo(a)pyrene (BaP) intermediate accumulation in mouse aortic endothelial cells (MAECs). Endoplasmic reticulum (ER) is the most active organelle involved in BaP metabolism. To examine the involvement of ER in catalase-induced BaP detoxification, we compared the level and distribution of XMEs, and the profile of BaP intermediates in the microsomes of wild-type and catalase transgenic endothelial cells. Our data showed that endothelial microsomes were enriched in cytochrome P450 (CYP) 1A1, CYP1B1 and epoxide hydrolase 1 (EH1), and contained considerable levels of quinone oxidoreductase-1 (NQO1) and glutathione S-transferase-pi (GSTP). Treatment of wild-type MAECs with 1μM BaP for 2 h increased the expression of microsomal CYP1A1, 1B1 and NQO1 by ~300, 64 and 116%, respectively. However, the same treatment did not significantly alter the expression of EH1 and GSTP. Overexpression of catalase did not significantly increase EH1, but upregulated BaP-induced expression of microsomal CYP1A1, 1B1, NQO1 and GSTP in the following order: 1A1>NQO1>GSTP>1B1. Overexpression of catalase did not alter the distribution of each of these enzymes in the microsomes. In contrast to our previous report showing lower level of BaP phenols versus BaP diols/diones in the whole-cell, this report demonstrated that the sum of microsomal BaP phenolic metabolites were ~60% greater than that of the BaP diols/diones after exposure of microsomes to BaP. Overexpression of catalase reduced the concentrations of microsomal BaP phenols and diols/diones by ~45 and 95%, respectively. This process enhanced the ratio of BaP phenol versus diol/dione metabolites in a potent manner. Taken together, upregulation of phase II XMEs and CYP1 proteins, but not EH1 in the ER might be the mechanism by which overexpression of catalase reduces the levels of all the BaP metabolites, and

Prenatal exposure to an NMDA receptor antagonist, MK-801 reduces density of parvalbumin-immunoreactive GABAergic neurons in the medial prefrontal cortex and enhances phencyclidine-induced hyperlocomotion but not behavioral sensitization to methamphetamine in postpubertal rats.

Science.gov (United States)

Abekawa, Tomohiro; Ito, Koki; Nakagawa, Shin; Koyama, Tsukasa

2007-06-01

Neurodevelopmental deficits of parvalbumin-immunoreactive gamma-aminobutyric acid (GABA)ergic interneurons in prefrontal cortex have been reported in schizophrenia. Glutamate influences the proliferation of this type of interneuron by an N-methyl-D-aspartate (NMDA)-receptor-mediated mechanism. The present study hypothesized that prenatal blockade of NMDA receptors would disrupt GABAergic neurodevelopment, resulting in differences in effects on behavioral responses to a noncompetitive NMDA antagonist, phencyclidine (PCP), and a dopamine releaser, methamphetamine (METH). GABAergic neurons were immunohistochemically stained with parvalbumin antibody. Psychostimulant-induced hyperlocomotion was measured using an infrared sensor. Prenatal exposure (E15-E18) to the NMDA receptor antagonist MK-801 reduced the density of parvalbumin-immunoreactive neurons in rat medial prefrontal cortex on postnatal day 63 (P63) and enhanced PCP-induced hyperlocomotion but not the acute effects of METH on P63 or the development of behavioral sensitization. Prenatal exposure to MK-801 reduced the number of parvalbumin-immunoreactive neurons even on postnatal day 35 (P35) and did not enhance PCP-induced hyperlocomotion, the acute effects of METH on P35, or the development of behavioral sensitization to METH. These findings suggest that prenatal blockade of NMDA receptors disrupts GABAergic neurodevelopment in medial prefrontal cortex, and that this disruption of GABAergic development may be related to the enhancement of the locomotion-inducing effect of PCP in postpubertal but not juvenile offspring. GABAergic deficit is unrelated to the effects of METH. This GABAergic neurodevelopmental disruption and the enhanced PCP-induced hyperlocomotion in adult offspring prenatally exposed to MK-801 may prove useful as a new model of the neurodevelopmental process of pathogenesis of treatment-resistant schizophrenia via an NMDA-receptor-mediated hypoglutamatergic mechanism.

Development of a Hypoallergenic Recombinant Parvalbumin for First-in-Man Subcutaneous Immunotherapy of Fish Allergy

DEFF Research Database (Denmark)

Zuidmeer-Jongejan, Laurian; Huber, Hans; Swoboda, Ines

2015-01-01

BACKGROUND: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. OBJECTIVES: Preclinical characterization and good manufacturing practice (GMP) production...... chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural...... as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human...

Pressure-temperature stability, Ca2+ binding, and pressure-temperature phase diagram of cod parvalbumin: Gad m 1.

Science.gov (United States)

Somkuti, Judit; Bublin, Merima; Breiteneder, Heimo; Smeller, László

2012-07-31

Fish allergy is associated with IgE-mediated hypersensitivity reactions to parvalbumins, which are small calcium-binding muscle proteins and represent the major and sole allergens for 95% of fish-allergic patients. We performed Fourier transform infrared and tryptophan fluorescence spectroscopy to explore the pressure-temperature (p-T) phase diagram of cod parvalbumin (Gad m 1) and to elucidate possible new ways of pressure-temperature inactivation of this food allergen. Besides the secondary structure of the protein, the Ca(2+) binding to aspartic and glutamic acid residues was detected. The phase diagram was found to be quite complex, containing partially unfolded and molten globule states. The Ca(2+) ions were essential for the formation of the native structure. A molten globule conformation appears at 50 °C and atmospheric pressure, which converts into an unordered aggregated state at 75 °C. At >200 MPa, only heat unfolding, but no aggregation, was observed. A pressure of 500 MPa leads to a partially unfolded state at 27 °C. The complete pressure unfolding could only be reached at an elevated temperature (40 °C) and pressure (1.14 GPa). A strong correlation was found between Ca(2+) binding and the protein conformation. The partially unfolded state was reversibly refolded. The completely unfolded molecule, however, from which Ca(2+) was released, could not refold. The heat-unfolded protein was trapped either in the aggregated state or in the molten globule state without aggregation at elevated pressures. The heat-treated and the combined heat- and pressure-treated protein samples were tested with sera of allergic patients, but no change in allergenicity was found.

Oxidized SOD1 alters proteasome activities in vitro and in the cortex of SOD1 overexpressing mice.

Science.gov (United States)

Le Pecheur, Marie; Bourdon, Emmanuel; Paly, Evelyne; Farout, Luc; Friguet, Bertrand; London, Jacqueline

2005-07-04

Premature ageing, one of the characteristics of Down syndrome (DS), may involve oxidative stress and impairment of proteasome activity. Transgenic mice overexpressing the human copper/zinc superoxide dismutase (SOD1) gene are one of the first murine models for DS and it has been shown that SOD1 overexpression might be either deleterious or beneficial. Here, we show a reduction in proteasome activities in the cortex of SOD1 transgenic mice and an associated increase in the content of oxidized SOD1 protein. As we demonstrate that in vitro oxidized SOD can inhibit purified proteasome peptidase activities, modified SOD1 might be partially responsible for proteasome inhibition shown in SOD1 transgenic mice.

Docosahexaenoic Acid Modulates a HER2-Associated Lipogenic Phenotype, Induces Apoptosis, and Increases Trastuzumab Action in HER2-Overexpressing Breast Carcinoma Cells

OpenAIRE

Ravacci, Graziela Rosa; Brentani, Maria Mitzi; Tortelli, Tharcisio Citrângulo; Torrinhas, Raquel Suzana M. M.; Santos, Jéssica Reis; Logullo, Angela Flávia; Waitzberg, Dan Linetzky

2015-01-01

In breast cancer, lipid metabolic alterations have been recognized as potential oncogenic stimuli that may promote malignancy. To investigate whether the oncogenic nature of lipogenesis closely depends on the overexpression of HER2 protooncogene, the normal breast cell line, HB4a, was transfected with HER2 cDNA to obtain HER2-overexpressing HB4aC5.2 cells. Both cell lines were treated with trastuzumab and docosahexaenoic acid. HER2 overexpression was accompanied by an increase in the expressi...

Overexpression of cerebral and hepatic cytochrome P450s alters behavioral activity of rat offspring following prenatal exposure to lindane

Energy Technology Data Exchange (ETDEWEB)

Johri, Ashu; Yadav, Sanjay; Dhawan, Alok [Developmental Toxicology Division, Industrial Toxicology Research Centre, P. O. Box 80, M. G. Marg, Lucknow-226 001, U. P. (India); Parmar, Devendra [Developmental Toxicology Division, Industrial Toxicology Research Centre, P. O. Box 80, M. G. Marg, Lucknow-226 001, U. P. (India)

2007-12-15

Oral administration of different doses (0.0625, 0.125 or 0.25 mg/kg corresponding to 1/1400th, 1/700th or 1/350th of LD{sub 50}) of lindane to the pregnant Wistar rats from gestation days 5 to 21 were found to produce a dose-dependent increase in the activity of cytochrome P450 (CYP)-dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) in brain and liver of offspring postnatally at 3 weeks. The increase in the activity of CYP monooxygenases was found to be associated with the increase in the mRNA and protein expression of xenobiotic metabolizing CYP1A, 2B and 2E1 isoenzymes in the brain and liver of offspring. Dose-dependent alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 3 weeks have suggested that increase in CYP activity may possibly lead to the formation of metabolites to the levels that may be sufficient to alter the behavioral activity of the offspring. Interestingly, the inductive effect on cerebral and hepatic CYPs was found to persist postnatally up to 6 weeks in the offspring at the relatively higher doses (0.125 and 0.25 mg/kg) of lindane and up to 9 weeks at the highest dose (0.25 mg/kg), though the magnitude of induction was less than that observed at 3 weeks. Alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 6 and 9 weeks, though significant only in the offspring at 3 and 6-week of age, have further indicated that due to the reduced activity of the CYPs during the ontogeny, lindane and its metabolites may not be effectively cleared from the brain. The data suggest that low dose prenatal exposure to the pesticide has the potential to produce overexpression of xenobiotic metabolizing CYPs in brain and liver of the offspring which may account for the behavioral changes observed in the offspring.

Overexpression of cerebral and hepatic cytochrome P450s alters behavioral activity of rat offspring following prenatal exposure to lindane

International Nuclear Information System (INIS)

Johri, Ashu; Yadav, Sanjay; Dhawan, Alok; Parmar, Devendra

2007-01-01

Oral administration of different doses (0.0625, 0.125 or 0.25 mg/kg corresponding to 1/1400th, 1/700th or 1/350th of LD 50 ) of lindane to the pregnant Wistar rats from gestation days 5 to 21 were found to produce a dose-dependent increase in the activity of cytochrome P450 (CYP)-dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) in brain and liver of offspring postnatally at 3 weeks. The increase in the activity of CYP monooxygenases was found to be associated with the increase in the mRNA and protein expression of xenobiotic metabolizing CYP1A, 2B and 2E1 isoenzymes in the brain and liver of offspring. Dose-dependent alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 3 weeks have suggested that increase in CYP activity may possibly lead to the formation of metabolites to the levels that may be sufficient to alter the behavioral activity of the offspring. Interestingly, the inductive effect on cerebral and hepatic CYPs was found to persist postnatally up to 6 weeks in the offspring at the relatively higher doses (0.125 and 0.25 mg/kg) of lindane and up to 9 weeks at the highest dose (0.25 mg/kg), though the magnitude of induction was less than that observed at 3 weeks. Alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 6 and 9 weeks, though significant only in the offspring at 3 and 6-week of age, have further indicated that due to the reduced activity of the CYPs during the ontogeny, lindane and its metabolites may not be effectively cleared from the brain. The data suggest that low dose prenatal exposure to the pesticide has the potential to produce overexpression of xenobiotic metabolizing CYPs in brain and liver of the offspring which may account for the behavioral changes observed in the offspring

Lack of parvalbumin in mice leads to behavioral deficits relevant to all human autism core symptoms and related neural morphofunctional abnormalities.

Science.gov (United States)

Wöhr, M; Orduz, D; Gregory, P; Moreno, H; Khan, U; Vörckel, K J; Wolfer, D P; Welzl, H; Gall, D; Schiffmann, S N; Schwaller, B

2015-03-10

Gene mutations and gene copy number variants are associated with autism spectrum disorders (ASDs). Affected gene products are often part of signaling networks implicated in synapse formation and/or function leading to alterations in the excitation/inhibition (E/I) balance. Although the network of parvalbumin (PV)-expressing interneurons has gained particular attention in ASD, little is known on PV's putative role with respect to ASD. Genetic mouse models represent powerful translational tools for studying the role of genetic and neurobiological factors underlying ASD. Here, we report that PV knockout mice (PV(-/-)) display behavioral phenotypes with relevance to all three core symptoms present in human ASD patients: abnormal reciprocal social interactions, impairments in communication and repetitive and stereotyped patterns of behavior. PV-depleted mice also showed several signs of ASD-associated comorbidities, such as reduced pain sensitivity and startle responses yet increased seizure susceptibility, whereas no evidence for behavioral phenotypes with relevance to anxiety, depression and schizophrenia was obtained. Reduced social interactions and communication were also observed in heterozygous (PV(+/-)) mice characterized by lower PV expression levels, indicating that merely a decrease in PV levels might be sufficient to elicit core ASD-like deficits. Structural magnetic resonance imaging measurements in PV(-/-) and PV(+/-) mice further revealed ASD-associated developmental neuroanatomical changes, including transient cortical hypertrophy and cerebellar hypoplasia. Electrophysiological experiments finally demonstrated that the E/I balance in these mice is altered by modification of both inhibitory and excitatory synaptic transmission. On the basis of the reported changes in PV expression patterns in several, mostly genetic rodent models of ASD, we propose that in these models downregulation of PV might represent one of the points of convergence, thus providing a

Significance of Aurora B overexpression in hepatocellular carcinoma. Aurora B Overexpression in HCC

International Nuclear Information System (INIS)

Lin, Zhong-Zhe; Jeng, Yung-Ming; Hu, Fu-Chang; Pan, Hung-Wei; Tsao, Hsin-Wei; Lai, Po-Lin; Lee, Po-Huang; Cheng, Ann-Lii; Hsu, Hey-Chi

2010-01-01

To investigate the significance of Aurora B expression in hepatocellular carcinoma (HCC). The Aurora B and Aurora A mRNA level was measured in 160 HCCs and the paired nontumorous liver tissues by reverse transcription-polymerase chain reaction. Mutations of the p53 and β-catenin genes were analyzed in 134 and 150 tumors, respectively, by direct sequencing of exon 2 to exon 11 of p53 and exon 3 of β-catenin. Anticancer effects of AZD1152-HQPA, an Aurora B kinase selective inhibitor, were examined in Huh-7 and Hep3B cell lines. Aurora B was overexpressed in 98 (61%) of 160 HCCs and in all 7 HCC cell lines examined. The overexpression of Aurora B was associated with Aurora A overexpression (P = 0.0003) and p53 mutation (P = 0.002) and was inversely associated with β-catenin mutation (P = 0.002). Aurora B overexpression correlated with worse clinicopathologic characteristics. Multivariate analysis confirmed that Aurora B overexpression was an independent poor prognostic factor, despite its interaction with Aurora A overexpression and mutations of p53 and β-catenin. In Huh-7 and Hep3B cells, AZD1152-HQPA induced proliferation blockade, histone H3 (Ser10) dephosphorylation, cell cycle disturbance, and apoptosis. Aurora B overexpression is an independent molecular marker predicting tumor invasiveness and poor prognosis of HCC. Aurora B kinase selective inhibitors are potential therapeutic agents for HCC treatment

Aryl hydrocarbon receptor overexpression in miniaturized follicles in female pattern hair loss.

Science.gov (United States)

Ramos, Paulo Müller; Brianezi, Gabrielli; Martins, Ana Carolina Pereira; Silva, Márcia Guimarães da; Marques, Mariângela Esther Alencar; Miot, Hélio Amante

2017-01-01

The etiopathogenesis of female pattern hair loss is still poorly understood. In addition to genetic and hormonal elements, environmental factors could be involved. The aryl hydrocarbon receptor is expressed in keratinocytes and can be activated by environmental pollutants leading to alterations in the cell cycle, inflammation, and apoptosis. Here we demonstrate the overexpression of nuclear aryl hydrocarbon receptors in miniaturized hair follicles in female pattern hair loss.

Overexpression of GbWRKY1 positively regulates the Pi starvation response by alteration of auxin sensitivity in Arabidopsis.

Science.gov (United States)

Xu, Li; Jin, Li; Long, Lu; Liu, Linlin; He, Xin; Gao, Wei; Zhu, Longfu; Zhang, Xianlong

2012-12-01

Overexpression of a cotton defense-related gene GbWRKY1 in Arabidopsis resulted in modification of the root system by enhanced auxin sensitivity to positively regulate the Pi starvation response. GbWRKY1 was a cloned WRKY transcription factor from Gossypium barbadense, which was firstly identified as a defense-related gene and showed moderate similarity with AtWRKY75 from Arabidopsis thaliana. Overexpression of GbWRKY1 in Arabidopsis resulted in attenuated Pi starvation stress symptoms, including reduced accumulation of anthocyanin and impaired density of lateral roots (LR) in low Pi stress. The study also indicated that overexpression of GbWRKY1 caused plants constitutively exhibited Pi starvation response including increased development of LR, relatively high level of total P and Pi, high expression level of some high-affinity Pi transporters and phosphatases as well as enhanced accumulation of acid phosphatases activity during Pi-sufficient. It was speculated that GbWRKY1 may act as a positive regulator in the Pi starvation response as well as AtWRKY75. GbWRKY1 probably involves in the modulation of Pi homeostasis and participates in the Pi allocation and remobilization but do not accumulate more Pi in Pi-deficient condition, which was different from the fact that AtWRKY75 influenced the Pi status of the plant during Pi deprivation by increasing root surface area and accumulation of more Pi. Otherwise, further study suggested that the overexpression plants were more sensitive to auxin than wild-type and GbWRKY1 may partly influence the LPR1-dependent (low phosphate response 1) Pi starvation signaling pathway and was putatively independent of SUMO E3 ligase SIZ1 and PHR1 (phosphate starvation response 1) in response to Pi starvation.

Selective expression of KCNS3 potassium channel α-subunit in parvalbumin-containing GABA neurons in the human prefrontal cortex.

Directory of Open Access Journals (Sweden)

Danko Georgiev

Full Text Available The cognitive deficits of schizophrenia appear to be associated with altered cortical GABA neurotransmission in the subsets of inhibitory neurons that express either parvalbumin (PV or somatostatin (SST. Identification of molecular mechanisms that operate selectively in these neurons is essential for developing targeted therapeutic strategies that do not influence other cell types. Consequently, we sought to identify, in the human cortex, gene products that are expressed selectively by PV and/or SST neurons, and that might contribute to their distinctive functional properties. Based on previously reported expression patterns in the cortex of mice and humans, we selected four genes: KCNS3, LHX6, KCNAB1, and PPP1R2, encoding K(+ channel Kv9.3 modulatory α-subunit, LIM homeobox protein 6, K(+ channel Kvβ1 subunit, and protein phosphatase 1 regulatory subunit 2, respectively, and examined their colocalization with PV or SST mRNAs in the human prefrontal cortex using dual-label in situ hybridization with (35S- and digoxigenin-labeled antisense riboprobes. KCNS3 mRNA was detected in almost all PV neurons, but not in SST neurons, and PV mRNA was detected in >90% of KCNS3 mRNA-expressing neurons. LHX6 mRNA was detected in almost all PV and >90% of SST neurons, while among all LHX6 mRNA-expressing neurons 50% expressed PV mRNA and >44% expressed SST mRNA. KCNAB1 and PPP1R2 mRNAs were detected in much larger populations of cortical neurons than PV or SST neurons. These findings indicate that KCNS3 is a selective marker of PV neurons, whereas LHX6 is expressed by both PV and SST neurons. KCNS3 and LHX6 might be useful for characterizing cell-type specific molecular alterations of cortical GABA neurotransmission and for the development of novel treatments targeting PV and/or SST neurons in schizophrenia.

Induction of epigenetic variation in Arabidopsis by over-expression of DNA METHYLTRANSFERASE1 (MET1.

Directory of Open Access Journals (Sweden)

Samuel Brocklehurst

Full Text Available Epigenetic marks such as DNA methylation and histone modification can vary among plant accessions creating epi-alleles with different levels of expression competence. Mutations in epigenetic pathway functions are powerful tools to induce epigenetic variation. As an alternative approach, we investigated the potential of over-expressing an epigenetic function, using DNA METHYLTRANSFERASE1 (MET1 for proof-of-concept. In Arabidopsis thaliana, MET1 controls maintenance of cytosine methylation at symmetrical CG positions. At some loci, which contain dense DNA methylation in CG- and non-CG context, loss of MET1 causes joint loss of all cytosines methylation marks. We find that over-expression of both catalytically active and inactive versions of MET1 stochastically generates new epi-alleles at loci encoding transposable elements, non-coding RNAs and proteins, which results for most loci in an increase in expression. Individual transformants share some common phenotypes and genes with altered gene expression. Altered expression states can be transmitted to the next generation, which does not require the continuous presence of the MET1 transgene. Long-term stability and epigenetic features differ for individual loci. Our data show that over-expression of MET1, and potentially of other genes encoding epigenetic factors, offers an alternative strategy to identify epigenetic target genes and to create novel epi-alleles.

Overexpression of Drosophila frataxin triggers cell death in an iron-dependent manner.

Science.gov (United States)

Edenharter, Oliver; Clement, Janik; Schneuwly, Stephan; Navarro, Juan A

2017-12-01

Friedreich ataxia (FRDA) is the most important autosomal recessive ataxia in the Caucasian population. FRDA patients display severe neurological and cardiac symptoms that reflect a strong cellular and axonal degeneration. FRDA is caused by a loss of function of the mitochondrial protein frataxin which impairs the biosynthesis of iron-sulfur clusters and in turn the catalytic activity of several enzymes in the Krebs cycle and the respiratory chain leading to a diminished energy production. Although FRDA is due to frataxin depletion, overexpression might also be very helpful to better understand cellular functions of frataxin. In this work, we have increased frataxin expression in neurons to elucidate specific roles that frataxin might play in these tissues. Using molecular, biochemical, histological and behavioral methods, we report that frataxin overexpression is sufficient to increase oxidative phosphorylation, modify mitochondrial morphology, alter iron homeostasis and trigger oxidative stress-dependent cell death. Interestingly, genetic manipulation of mitochondrial iron metabolism by silencing mitoferrin successfully improves cell survival under oxidative-attack conditions, although enhancing antioxidant defenses or mitochondrial fusion failed to ameliorate frataxin overexpression phenotypes. This result suggests that cell degeneration is directly related to enhanced incorporation of iron into the mitochondria. Drosophila frataxin overexpression might also provide an alternative approach to identify processes that are important in FRDA such as changes in mitochondrial morphology and oxidative stress induced cell death.

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis

Science.gov (United States)

Watanabe, Miki; Muraleedharan, Ranjithmenon; Lambert, Paul F.; Lane, Andrew N.; Romick-Rosendale, Lindsey E.; Wells, Susanne I.

2017-01-01

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth. PMID:28558019

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis.

Directory of Open Access Journals (Sweden)

Marie C Matrka

Full Text Available The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos. To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth.

P53 overexpression and outcome of radiation therapy in head and neck cancers

International Nuclear Information System (INIS)

Kim, In Ah; Choi, Ihl Bhong; Kang, Ki Mun; Jang, Ji Young; Kim, Kyung Mi; Park, Kyung Shin; Kim, Young Shin; Kang, Chang Suk; Cho, Seung Ho; Kim, Hyung Tae

1999-01-01

Experimental studies have implicated the wild type p53 in cellular response to radiation. Whether altered p53 function can lead to changes in clinical radiocurability remains an area of ongoing study. This study was performed to investigate whether any correlation between change of p53 and outcome of curative radiation therapy in patients with head and neck cancers. Immunohistochemical analysis with a mouse monoclonal antibody (D0-7) specific for human p53 was used to detect to overexpression of protein in formalin fixed, paraffin-embedded tumor sample from 55 head and neck cancer patients treated with curative radiation therapy (median dose of 7020 cGy) from February 1988 to March 1996 at St. Mary's Hospital. Overexpression of p53 was correlated with locoregional control and survival using Kaplan-Meier method. A Cox regression multivariate analysis was performed that included all clinical variables and status of p53 expression. Thirty-seven (67.2%) patients showed overexpression of p53 by immunohistochemical staining in their tumor. One hundred percent of oral cavity, 76% of laryngeal, 66.7% of oropharyngeal, 66.7% of hypopharyngeal cancer showed p53 overexpression (p=0.05). The status of p53 had significant relationship with stage of disease (p=0.03) and history of smoking (p=0.001). The overexpression of p53 was not predictive of response rate to radiation therapy. The locoregional control was not significantly affected by p53 status. Overexpression of p53 didn't have any prognostic implication for disease free survival and overall survival. Primary site and stage of disease were significant prognostic factors for survival. The p53 overexpression as detected by immunohistochemical staining had significant correlation with stage, primary site of disease and smoking habit of patients. The p53 overexpression didn't have any predictive value for outcome of curative radiation therapy in a group of head and neck cancers

P53 overexpression and outcome of radiation therapy in head and neck cancers

Energy Technology Data Exchange (ETDEWEB)

Kim, In Ah; Choi, Ihl Bhong; Kang, Ki Mun; Jang, Ji Young; Kim, Kyung Mi; Park, Kyung Shin; Kim, Young Shin; Kang, Chang Suk; Cho, Seung Ho; Kim, Hyung Tae [College of Medicine, The Catholic Univ., Seoul (Korea, Republic of)

1999-03-01

Experimental studies have implicated the wild type p53 in cellular response to radiation. Whether altered p53 function can lead to changes in clinical radiocurability remains an area of ongoing study. This study was performed to investigate whether any correlation between change of p53 and outcome of curative radiation therapy in patients with head and neck cancers. Immunohistochemical analysis with a mouse monoclonal antibody (D0-7) specific for human p53 was used to detect to overexpression of protein in formalin fixed, paraffin-embedded tumor sample from 55 head and neck cancer patients treated with curative radiation therapy (median dose of 7020 cGy) from February 1988 to March 1996 at St. Mary's Hospital. Overexpression of p53 was correlated with locoregional control and survival using Kaplan-Meier method. A Cox regression multivariate analysis was performed that included all clinical variables and status of p53 expression. Thirty-seven (67.2%) patients showed overexpression of p53 by immunohistochemical staining in their tumor. One hundred percent of oral cavity, 76% of laryngeal, 66.7% of oropharyngeal, 66.7% of hypopharyngeal cancer showed p53 overexpression (p=0.05). The status of p53 had significant relationship with stage of disease (p=0.03) and history of smoking (p=0.001). The overexpression of p53 was not predictive of response rate to radiation therapy. The locoregional control was not significantly affected by p53 status. Overexpression of p53 didn't have any prognostic implication for disease free survival and overall survival. Primary site and stage of disease were significant prognostic factors for survival. The p53 overexpression as detected by immunohistochemical staining had significant correlation with stage, primary site of disease and smoking habit of patients. The p53 overexpression didn't have any predictive value for outcome of curative radiation therapy in a group of head and neck cancers.

Overexpression of soluble ADAM33 promotes a hypercontractile phenotype of the airway smooth muscle cell in rat

Energy Technology Data Exchange (ETDEWEB)

Duan, Yiyuan; Long, Jiaoyue; Chen, Jun; Jiang, Xuemei; Zhu, Jian; Jin, Yang; Lin, Feng; Zhong, Jun; Xu, Rong [Key Laboratory of Biorheological Science and Technology, Ministry of Education, and Bioengineering College, Chongqing University, Shapingba, Chongqing 400030 (China); Mao, Lizheng [Jiangsu Asialand Biomed-Technology Co. Ltd., Changzhou, Jiangsu 213164 (China); Deng, Linhong, E-mail: dlh@cczu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, and Bioengineering College, Chongqing University, Shapingba, Chongqing 400030 (China); Changzhou Key Laboratory of Respiratory Medical Engineering, Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, Jiangsu 213164 (China)

2016-11-15

A disintegrin and metalloproteinase 33 (ADAM33) has been identified as a susceptibility gene for asthma, but details of the causality are not fully understood. We hypothesize that soluble ADAM33 (sADAM33) overexpression can alter the mechanical behaviors of airway smooth muscle cells (ASMCs) via regulation of the cell's contractile phenotype, and thus contributes to airway hyperresponsiveness (AHR) in asthma. To test this hypothesis, we either overexpressed or knocked down the sADAM33 in rat ASMCs by transfecting the cells with sADAM33 coding sequence or a small interfering RNA (siRNA) that specifically targets the ADAM33 disintegrin domain, and subsequently assessed the cells for stiffness, contractility and traction force, together with the expression level of contractile and proliferative phenotype markers. We also investigated whether these changes were dependent on Rho/ROCK pathway by culturing the ASMCs either in the absence or presence of ROCK inhibitor (H1152). The results showed that the ASMCs with sADAM33 overexpression were stiffer and more contractile, generated greater traction force, exhibited increased expression levels of contractile phenotype markers and markedly enhanced Rho activation. Furthermore these changes were largely attenuated when the cells were cultured in the presence of H-1152. However, the knock-down of ADAM33 seemed insufficient to influence majority of the mechanical behaviors of the ASMCs. Taken together, we demonstrated that sADAM33 overexpression altered the mechanical behaviors of ASMCs in vitro, which was most likely by promoting a hypercontractile phenotype transition of ASMCs through Rho/ROCK pathway. This revelation may establish the previously missing link between ADAM33 expression and AHR, and also provide useful insight for targeting sADAM33 in asthma prevention and therapy. - Highlights: • sADAM33 overexpression enhances the stiffness, traction force and contractility of ASMCs. • sADAM33 overexpression promotes

Overexpression of soluble ADAM33 promotes a hypercontractile phenotype of the airway smooth muscle cell in rat

International Nuclear Information System (INIS)

Duan, Yiyuan; Long, Jiaoyue; Chen, Jun; Jiang, Xuemei; Zhu, Jian; Jin, Yang; Lin, Feng; Zhong, Jun; Xu, Rong; Mao, Lizheng; Deng, Linhong

2016-01-01

A disintegrin and metalloproteinase 33 (ADAM33) has been identified as a susceptibility gene for asthma, but details of the causality are not fully understood. We hypothesize that soluble ADAM33 (sADAM33) overexpression can alter the mechanical behaviors of airway smooth muscle cells (ASMCs) via regulation of the cell's contractile phenotype, and thus contributes to airway hyperresponsiveness (AHR) in asthma. To test this hypothesis, we either overexpressed or knocked down the sADAM33 in rat ASMCs by transfecting the cells with sADAM33 coding sequence or a small interfering RNA (siRNA) that specifically targets the ADAM33 disintegrin domain, and subsequently assessed the cells for stiffness, contractility and traction force, together with the expression level of contractile and proliferative phenotype markers. We also investigated whether these changes were dependent on Rho/ROCK pathway by culturing the ASMCs either in the absence or presence of ROCK inhibitor (H1152). The results showed that the ASMCs with sADAM33 overexpression were stiffer and more contractile, generated greater traction force, exhibited increased expression levels of contractile phenotype markers and markedly enhanced Rho activation. Furthermore these changes were largely attenuated when the cells were cultured in the presence of H-1152. However, the knock-down of ADAM33 seemed insufficient to influence majority of the mechanical behaviors of the ASMCs. Taken together, we demonstrated that sADAM33 overexpression altered the mechanical behaviors of ASMCs in vitro, which was most likely by promoting a hypercontractile phenotype transition of ASMCs through Rho/ROCK pathway. This revelation may establish the previously missing link between ADAM33 expression and AHR, and also provide useful insight for targeting sADAM33 in asthma prevention and therapy. - Highlights: • sADAM33 overexpression enhances the stiffness, traction force and contractility of ASMCs. • sADAM33 overexpression promotes a

Chemokine receptors and cortical interneuron dysfunction in schizophrenia.

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Volk, David W; Chitrapu, Anjani; Edelson, Jessica R; Lewis, David A

2015-09-01

Alterations in inhibitory (GABA) neurons, including deficiencies in the GABA synthesizing enzyme GAD67, in the prefrontal cortex in schizophrenia are pronounced in the subpopulations of neurons that contain the calcium-binding protein parvalbumin or the neuropeptide somatostatin. The presence of similar illness-related deficits in the transcription factor Lhx6, which regulates prenatal development of parvalbumin and somatostatin neurons, suggests that cortical GABA neuron dysfunction may be related to disturbances in utero. Since the chemokine receptors CXCR4 and CXCR7 guide the migration of cortical parvalbumin and somatostatin neurons from their birthplace in the medial ganglionic eminence to their final destination in the neocortex, we sought to determine whether altered CXCR4 and/or CXCR7 mRNA levels were associated with disturbances in GABA-related markers in schizophrenia. Quantitative PCR was used to quantify CXCR4 and CXCR7 mRNA levels in the prefrontal cortex of 62 schizophrenia and 62 healthy comparison subjects that were previously characterized for markers of parvalbumin and somatostatin neurons and in antipsychotic-exposed monkeys. We found elevated mRNA levels for CXCR7 (+29%; pschizophrenia subjects but not in antipsychotic-exposed monkeys. CXCR7 mRNA levels were inversely correlated with mRNA levels for GAD67, parvalbumin, somatostatin, and Lhx6 in schizophrenia but not in healthy subjects. These findings suggest that higher mRNA levels for CXCR7, and possibly CXCR4, may represent a compensatory mechanism to sustain the migration and correct positioning of cortical parvalbumin and somatostatin neurons in the face of other insults that disrupt the prenatal development of cortical GABA neurons in schizophrenia. Copyright © 2014 Elsevier B.V. All rights reserved.

Modulation of ethylene responses by OsRTH1 overexpression reveals the biological significance of ethylene in rice seedling growth and development

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Zhang, Wei; Zhou, Xin; Wen, Chi-Kuang

2012-01-01

Overexpression of Arabidopsis Reversion-To-ethylene Sensitivity1 (RTE1) results in whole-plant ethylene insensitivity dependent on the ethylene receptor gene Ethylene Response1 (ETR1). However, overexpression of the tomato RTE1 homologue Green Ripe (GR) delays fruit ripening but does not confer whole-plant ethylene insensitivity. It was decided to investigate whether aspects of ethylene-induced growth and development of the monocotyledonous model plant rice could be modulated by rice RTE1 homologues (OsRTH genes). Results from a cross-species complementation test in Arabidopsis showed that OsRTH1 overexpression complemented the rte1-2 loss-of-function mutation and conferred whole-plant ethylene insensitivity in an ETR1-dependent manner. In contrast, OsRTH2 and OsRTH3 overexpression did not complement rte1-2 or confer ethylene insensitivity. In rice, OsRTH1 overexpression substantially prevented ethylene-induced alterations in growth and development, including leaf senescence, seedling leaf elongation and development, coleoptile elongation or curvature, and adventitious root development. Results of subcellular localizations of OsRTHs, each fused with the green fluorescent protein, in onion epidermal cells suggested that the three OsRTHs were predominantly localized to the Golgi. OsRTH1 may be an RTE1 orthologue of rice and modulate rice ethylene responses. The possible roles of auxins and gibberellins in the ethylene-induced alterations in growth were evaluated and the biological significance of ethylene in the early stage of rice seedling growth is discussed. PMID:22451723

HDAC2 expression in parvalbumin interneurons regulates synaptic plasticity in the mouse visual cortex

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Alexi Nott

2015-01-01

Full Text Available An experience-dependent postnatal increase in GABAergic inhibition in the visual cortex is important for the closure of a critical period of enhanced synaptic plasticity. Although maturation of the subclass of parvalbumin (Pv–expressing GABAergic interneurons is known to contribute to critical period closure, the role of epigenetics on cortical inhibition and synaptic plasticity has not been explored. The transcription regulator, histone deacetylase 2 (HDAC2, has been shown to modulate synaptic plasticity and learning processes in hippocampal excitatory neurons. We found that genetic deletion of HDAC2 specifically from Pv interneurons reduces inhibitory input in the visual cortex of adult mice and coincides with enhanced long-term depression that is more typical of young mice. These findings show that HDAC2 loss in Pv interneurons leads to a delayed closure of the critical period in the visual cortex and supports the hypothesis that HDAC2 is a key negative regulator of synaptic plasticity in the adult brain.

HDAC2 expression in parvalbumin interneurons regulates synaptic plasticity in the mouse visual cortex.

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Nott, Alexi; Cho, Sukhee; Seo, Jinsoo; Tsai, Li-Huei

2015-01-01

An experience-dependent postnatal increase in GABAergic inhibition in the visual cortex is important for the closure of a critical period of enhanced synaptic plasticity. Although maturation of the subclass of Parvalbumin (Pv)-expressing GABAergic interneurons is known to contribute to critical period closure, the role of epigenetics on cortical inhibition and synaptic plasticity has not been explored. The transcription regulator, histone deacetylase 2 (HDAC2), has been shown to modulate synaptic plasticity and learning processes in hippocampal excitatory neurons. We found that genetic deletion of HDAC2 specifically from Pv-interneurons reduces inhibitory input in the visual cortex of adult mice, and coincides with enhanced long-term depression (LTD) that is more typical of young mice. These findings show that HDAC2 loss in Pv-interneurons leads to a delayed closure of the critical period in the visual cortex and supports the hypothesis that HDAC2 is a key negative regulator of synaptic plasticity in the adult brain.

Acoustic Trauma Changes the Parvalbumin-Positive Neurons in Rat Auditory Cortex

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Congli Liu

2018-01-01

Full Text Available Acoustic trauma is being reported to damage the auditory periphery and central system, and the compromised cortical inhibition is involved in auditory disorders, such as hyperacusis and tinnitus. Parvalbumin-containing neurons (PV neurons, a subset of GABAergic neurons, greatly shape and synchronize neural network activities. However, the change of PV neurons following acoustic trauma remains to be elucidated. The present study investigated how auditory cortical PV neurons change following unilateral 1 hour noise exposure (left ear, one octave band noise centered at 16 kHz, 116 dB SPL. Noise exposure elevated the auditory brainstem response threshold of the exposed ear when examined 7 days later. More detectable PV neurons were observed in both sides of the auditory cortex of noise-exposed rats when compared to control. The detectable PV neurons of the left auditory cortex (ipsilateral to the exposed ear to noise exposure outnumbered those of the right auditory cortex (contralateral to the exposed ear. Quantification of Western blotted bands revealed higher expression level of PV protein in the left cortex. These findings of more active PV neurons in noise-exposed rats suggested that a compensatory mechanism might be initiated to maintain a stable state of the brain.

Overexpression of a SNARE protein AtBS14b alters BR response in Arabidopsis.

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Zhu, Zhong Xin; Ye, Hong Bo; Xuan, Yuan Hu; Yao, Da Nian

2014-12-01

N-ethyl-maleimide sensitive factor adaptor protein receptor (SNAREs) domain-containing proteins were known as key players in vesicle-associated membrane fusion. Genetic screening has revealed the function of SNAREs in different aspects of plant biology, but the role of many SNAREs are still unknown. In this study, we have characterized the role of Arabidopsis Qc-SNARE protein AtBS14b in brassinosteroids (BRs) signaling pathway. AtBS14b overexpression (AtBS14b ox) plants exhibited short hypocotyl and petioles lengths as well as insensitivity to exogenously supplied BR, while AtBS14b mutants did not show any visible BR-dependent morphological differences. BR biosynthesis enzyme BR6OX2 expression was slightly lower in AtBS14b ox than in wild type plants. Further BR-mediated repression of BR6OX2, CPD and DWF4 was inhibited in AtBS14b ox plants. AtBS14b-mCherry fusion protein localized in vesicular compartments surrounding plasma membrane in N. benthamiana leaves. In addition, isolation of AtBS14b-interacting BR signaling protein, which localized in plasma membrane, showed that AtBS14b directly interacted with membrane steroid binding protein 1 (MSBP1), but did not interact with BAK1 or BRI1. These data suggested that Qc-SNARE protein AtBS14b is the first SNARE protein identified that interacts with MSBP1, and the overexpression of AtBS14b modulates BR response in Arabidopsis.

Overexpression of Laccaria bicolor aquaporin JQ585595 alters root water transport properties in ectomycorrhizal white spruce (Picea glauca) seedlings.

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Xu, Hao; Kemppainen, Minna; El Kayal, Walid; Lee, Seong Hee; Pardo, Alejandro G; Cooke, Janice E K; Zwiazek, Janusz J

2015-01-01

The contribution of hyphae to water transport in ectomycorrhizal (ECM) white spruce (Picea glauca) seedlings was examined by altering expression of a major water-transporting aquaporin in Laccaria bicolor. Picea glauca was inoculated with wild-type (WT), mock transgenic or L. bicolor aquaporin JQ585595-overexpressing (OE) strains and exposed to root temperatures ranging from 5 to 20°C to examine the root water transport properties, physiological responses and plasma membrane intrinsic protein (PIP) expression in colonized plants. Mycorrhization increased shoot water potential, transpiration, net photosynthetic rates, root hydraulic conductivity and root cortical cell hydraulic conductivity in seedlings. At 20°C, OE plants had higher root hydraulic conductivity compared with WT plants and the increases were accompanied by higher expression of P. glauca PIP GQ03401_M18.1 in roots. In contrast to WT L. bicolor, the effects of OE fungi on root and root cortical cell hydraulic conductivities were abolished at 10 and 5°C in the absence of major changes in the examined transcript levels of P. glauca root PIPs. The results provide evidence for the importance of fungal aquaporins in root water transport of mycorrhizal plants. They also demonstrate links between hyphal water transport, root aquaporin expression and root water transport in ECM plants. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Gene Overexpression: Uses, Mechanisms, and Interpretation

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2012-01-01

The classical genetic approach for exploring biological pathways typically begins by identifying mutations that cause a phenotype of interest. Overexpression or misexpression of a wild-type gene product, however, can also cause mutant phenotypes, providing geneticists with an alternative yet powerful tool to identify pathway components that might remain undetected using traditional loss-of-function analysis. This review describes the history of overexpression, the mechanisms that are responsible for overexpression phenotypes, tests that begin to distinguish between those mechanisms, the varied ways in which overexpression is used, the methods and reagents available in several organisms, and the relevance of overexpression to human disease. PMID:22419077

Overexpression of Human-Derived DNMT3A Induced Intergenerational Inheritance of Active DNA Methylation Changes in Rat Sperm

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Xiaoguo Zheng

2017-12-01

Full Text Available DNA methylation is the major focus of studies on paternal epigenetic inheritance in mammals, but most previous studies about inheritable DNA methylation changes are passively induced by environmental factors. However, it is unclear whether the active changes mediated by variations in DNA methyltransferase activity are heritable. Here, we established human-derived DNMT3A (hDNMT3A transgenic rats to study the effect of hDNMT3A overexpression on the DNA methylation pattern of rat sperm and to investigate whether this actively altered DNA methylation status is inheritable. Our results revealed that hDNMT3A was overexpressed in the testis of transgenic rats and induced genome-wide alterations in the DNA methylation pattern of rat sperm. Among 5438 reliable loci identified with 64 primer-pair combinations using a methylation-sensitive amplification polymorphism method, 28.01% showed altered amplified band types. Among these amplicons altered loci, 68.42% showed an altered DNA methylation status in the offspring of transgenic rats compared with wild-type rats. Further analysis based on loci which had identical DNA methylation status in all three biological replicates revealed that overexpression of hDNMT3A in paternal testis induced hypermethylation in sperm of both genotype-negative and genotype-positive offspring. Among the differentially methylated loci, 34.26% occurred in both positive and negative offspring of transgenic rats, indicating intergenerational inheritance of active DNA methylation changes in the absence of hDNM3A transmission. Furthermore, 75.07% of the inheritable loci were hyper-methylated while the remaining were hypomethylated. Distribution analysis revealed that the DNA methylation variations mainly occurred in introns and intergenic regions. Functional analysis revealed that genes related to differentially methylated loci were involved in a wide range of functions. Finally, this study demonstrated that active DNA methylation

AAV-mediated overexpression of the CB1 receptor in the mPFC of adult rats alters cognitive flexibility, social behavior and emotional reactivity

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Matthias eKlugmann

2011-07-01

Full Text Available The endocannabinoid (ECB system is strongly involved in the regulation of cognitive processing and emotional behavior and evidence indicates that ECB signaling might affect these behavioral abilities by modulations of prefrontal cortical functions. The aim of the present study was to examine the role of the CB1 receptor in the medial prefrontal cortex (mPFC on cognitive flexibility and emotional behavior. Therefore, the CB1 receptor was overexpressed by adeno-associated virus (AAV vector-mediated gene transfer specifically in the mPFC of adult Wistar rats. Animals were then tested in different anxiety-related paradigms for emotional reactivity (e.g. elevated plus maze (EPM, light/dark emergence test (EMT, social interaction and the attentional set shift task (ASST - an adaptation of the human Wisconsin card sorting test - for cognitive abilities and behavioral flexibility. A subtle increase in exploratory behavior was found in CB1 receptor overexpressing animals (CB1-R compared to empty vector injected controls (Empty in the EMT and EPM, although general locomotor activity did not differ between the groups. During social interaction testing, social contact behavior towards the unknown conspecific was found to be decreased, whereas social withdrawal was increased in CB1-R animals and they showed an inadequate increase in exploratory behavior compared to control animals. In the ASST, impaired reversal learning abilities were detected in CB1-R animals compared to controls, indicating reduced behavioral flexibility. In conclusion, upregulation of the CB1 receptor specifically in the rat mPFC induces alterations in emotional reactivity, leads to inadequate social behavior and impairs cognitive flexibility. These findings might be relevant for neuropsychiatric disorders, since higher cortical CB1 receptor expression levels as well as similar behavioral impairments as observed in the present study have been described in schizophrenic patients.

Understanding Ion Binding Affinity and Selectivity in β-Parvalbumin Using Molecular Dynamics and Mean Spherical Approximation Theory.

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Kucharski, Amir N; Scott, Caitlin E; Davis, Jonathan P; Kekenes-Huskey, Peter M

2016-08-25

Parvalbumin (PV) is a globular calcium (Ca(2+))-selective protein expressed in a variety of biological tissues. Our computational studies of the rat β-parvalbumin (β-PV) isoform seek to elucidate the molecular thermodynamics of Ca(2+) versus magnesium (Mg(2+)) binding at the protein's two EF-hand motifs. Specifically, we have utilized molecular dynamics (MD) simulations and a mean-field electrolyte model (mean spherical approximation (MSA) theory) to delineate how the EF-hand scaffold controls the "local" thermodynamics of Ca(2+) binding selectivity over Mg(2+). Our MD simulations provide the probability density of metal-chelating oxygens within the EF-hand scaffolds for both Ca(2+) and Mg(2+), as well the conformational strain induced by Mg(2+) relative to Ca(2+) binding. MSA theory utilizes the binding domain oxygen and charge distributions to predict the chemical potential of ion binding, as well as their corresponding concentrations within the binding domain. We find that the electrostatic and steric contributions toward ion binding were similar for Mg(2+) and Ca(2+), yet the latter was 5.5 kcal/mol lower in enthalpy when internal strain within the EF hand was considered. We therefore speculate that beyond differences in dehydration energies for the Ca(2+) versus Mg(2+), strain induced in the β-PV EF hand by cation binding significantly contributes to the nearly 10,000-fold difference in binding affinity reported in the literature. We further complemented our analyses of local factors governing cation binding selectivity with whole-protein (global) contributions, such as interhelical residue-residue contacts and solvent exposure of hydrophobic surface. These contributions were found to be comparable for both Ca(2+)- and Mg(2+)-bound β-PV, which may implicate local factors, EF-hand strain, and dehydration, in providing the primary means of selectivity. We anticipate these methods could be used to estimate metal binding thermodynamics across a broad range of

Altering β-cell number through stable alteration of miR-21 and miR-34a expression

DEFF Research Database (Denmark)

Backe, Marie Balslev; Novotny, Guy Wayne; Christensen, Dan Ploug

2014-01-01

RNAs, miR-21 and miR-34a, may be involved in mediating cytokine-induced β-cell dysfunction. Therefore, manipulation of miR-21 and miR-34a levels may potentially be beneficial to β cells. To study the effect of long-term alterations of miR-21 or miR-34a levels upon net β-cell number, we stably overexpressed...

Overexpression of transforming growth factor-β1 in fetal monkey lung results in prenatal pulmonary fibrosis

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Tarantal, A.F.; Chen, H.; Shi, T.T.; Lu, C-H.; Fang, A.B.; Buckley, S.; Kolb, M.; Gauldie, J.; Warburton, D.; Shi, W.

2011-01-01

Altered transforming growth factor (TGF)-β expression levels have been linked to a variety of human respiratory diseases, including bronchopulmonary dysplasia and pulmonary fibrosis. However, a causative role for aberrant TGF-β in neonatal lung diseases has not been defined in primates. Exogenous and transient TGF-β1 overexpression in fetal monkey lung was achieved by transabdominal ultrasound-guided fetal intrapulmonary injection of adenoviral vector expressing TGF-β1 at the second or third trimester of pregnancy. The lungs were then harvested near term, and fixed for histology and immunohistochemistry. Lung hypoplasia was observed where TGF-β1 was overexpressed during the second trimester. The most clearly marked phenotype consisted of severe pulmonary and pleural fibrosis, which was independent of the gestational time point when TGF-β1 was overexpressed. Increased cell proliferation, particularly in α-smooth muscle actin-positive myofibroblasts, was detected within the fibrotic foci. But epithelium to mesenchyme transdifferentiation was not detected. Massive collagen fibres were deposited on the inner and outer sides of the pleural membrane, with an intact elastin layer in the middle. This induced fibrotic pathology persisted even after adenoviral-mediated TGF-β1 overexpression was no longer evident. Therefore, overexpression of TGF-β1 within developing fetal monkey lung results in severe and progressive fibrosis in lung parenchyma and pleural membrane, in addition to pulmonary hypoplasia. PMID:20351039

Effects of clusterin over-expression on metastatic progression and therapy in breast cancer

International Nuclear Information System (INIS)

Flanagan, Louise; Whyte, Lorna; Chatterjee, Namita; Tenniswood, Martin

2010-01-01

Clusterin is a secreted glycoprotein that is upregulated in a variety of cell lines in response to stress, and enhances cell survival. A second nuclear isoform of clusterin that is associated with cell death has also been identified. The aim of this study was to determine the role(s) of the secretory isoform in breast tumor progression and metastasis. To investigate the role of secretory clusterin in the biology of breast cancer tumor growth and resistance to therapy we have engineered an MCF-7 cell line (MCF-7CLU) that over-expresses clusterin. We have measured the in vitro effects of clusterin over-expression on cell cycle, cell death, and sensitivity to TNFalpha and tamoxifen. Using an orthotopic model of breast cancer, we have also determined the effects of over-expression of clusterin on tumor growth and metastatic progression. In vitro, over-expression of secretory clusterin alters the cell cycle kinetics and decreases the rate of cell death, resulting in the enhancement of cell growth. Over-expression of secretory clusterin also blocks the TNFalpha-mediated induction of p21 and abrogates the cleavage of Bax to t-Bax, rendering the MCF-7CLU cells significantly more resistant to the cytokine than the parental cells. Orthotopic primary tumors derived from MCF-7CLU cells grow significantly more rapidly than tumors derived from parental MCF-7 cells and, unlike the parental cells, metastasize frequently to the lungs. These data suggest that secretory clusterin, which is frequently up-regulated in breast cancers by common therapies, including anti-estrogens, may play a significant role in tumor growth, metastatic progression and subsequent drug resistance in surviving cells

Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

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Takabe, Piia; Bart, Geneviève; Ropponen, Antti; Rilla, Kirsi; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna

2015-01-01

Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma

Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

Energy Technology Data Exchange (ETDEWEB)

Takabe, Piia, E-mail: piia.takabe@uef.fi [University of Eastern Finland, Institute of Biomedicine, 70211 Kuopio (Finland); Bart, Geneviève [University of Eastern Finland, Institute of Biomedicine, 70211 Kuopio (Finland); Ropponen, Antti [University of Eastern Finland, Institute of Clinical Medicine, 70211 Kuopio (Finland); Rilla, Kirsi; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna [University of Eastern Finland, Institute of Biomedicine, 70211 Kuopio (Finland)

2015-09-10

Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma.

Escitalopram alters gene expression and HPA axis reactivity in rats following chronic overexpression of corticotropin-releasing factor from the central amygdala

Science.gov (United States)

Flandreau, Elizabeth I.; Bourke, Chase H.; Ressler, Kerry J.; Vale, Wylie W.; Nemeroff, Charles B.; Owens, Michael J.

2013-01-01

Summary We have previously demonstrated that viral-mediated overexpression of corticotropin-releasing factor (CRF) within the central nucleus of the amygdala (CeA) reproduces many of the behavioral and endocrine consequences of chronic stress. The present experiment sought to determine whether administration of the selective serotonin reuptake inhibitor (SSRI) escitalopram reverses the adverse effects of CeA CRF overexpression. In a 2 × 2 design, adult male rats received bilateral infusions of a control lentivirus or a lentivirus in which a portion of the CRF promoter is used to drive increased expression of CRF peptide. Four weeks later, rats were then implanted with an Alzet minipump to deliver vehicle or 10 mg/kg/day escitalopram for a 4-week period of time. The defensive withdrawal (DW) test of anxiety and the sucrose-preference test (SPT) of anhedonia were performed both before and after pump implantation. Additional post-implant behavioral tests included the elevated plus maze (EPM) and social interaction (SI) test. Following completion of behavioral testing, the dexamethasone/CRF test was performed to assess HPA axis reactivity. Brains were collected and expression of HPA axis-relevant transcripts were measured using in situ hybridization. Amygdalar CRF overexpression increased anxiety-like behavior in the DW test at week eight, which was only partially prevented by escitalopram. In both CRF-overexpressing and control groups, escitalopram decreased hippocampal CRF expression while increasing hypothalamic and hippocampal expression of the glucocorticoid receptor (GR). These gene expression changes were associated with a significant decrease in HPA axis reactivity in rats treated with escitalopram. Interestingly, escitalopram increased the rate of weight gain only in rats overexpressing CRF. Overall these data support our hypothesis that amygdalar CRF is critical in anxiety-like behavior; because the antidepressant was unable to reverse behavioral

Effects of CD44 and E-cadherin overexpression on the proliferation, adhesion and invasion of ovarian cancer cells.

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Mao, Meiya; Zheng, Xiaojiao; Jin, Bohong; Zhang, Fubin; Zhu, Linyan; Cui, Lining

2017-12-01

CD44 is a prognostic indicator of shorter survival time in ovarian cancer. E-cadherin fragmentation promotes the progression of ovarian cancer. However, the effects of CD44 and E-cadherin overexpression on ovarian cancer cells have remained elusive. The present study aimed to investigate the effects of overexpression of CD44 and E-cadherin on cell proliferation, adhesion and invasion of SKOV-3 and OVCAR-3 ovarian cancer cells. Overexpression of CD44 and E-cadherin was achieved by transfecting SKOV-3 and OVCAR-3 cells with viruses carrying the CD44 or E-cadherin gene, respectively. Expression of CD44 and E-cadherin was detected by western blot analysis. The proliferation of SKOV-3 and OVCAR-3 cells was measured by a Cell Counting Kit-8 at 0, 24 and 48 h after viral transfection. The adhesion ability of SKOV-3 and OVCAR-3 cells to the endothelial layer was detected. A Transwell invasion assay was utilized to assess the invasion ability of the cells. Overexpression of CD44 and E-cadherin in SKOV-3 and OVCAR-3 cells was confirmed by western blot. Compared with the blank or negative control groups, the CD44 overexpression groups of SKOV-3 and OVCAR-3 cells exhibited an increased cell proliferation rate at 24 and 48 h, whereas overexpression of E-cadherin did not alter the proliferation of these cells. Furthermore, compared with the blank and negative control groups, the cell adhesion and invasion ability in the CD44 overexpression groups of SKOV-3 and OVCAR-3 cells was markedly higher. There were no significant differences in adhesion ability between the E-cadherin overexpression group and the blank/negative control group. Of note, overexpression of E-cadherin decreased the invasive ability of SKOV-3 and OVCAR-3 cells. In conclusion, Overexpression of CD44 increased the proliferation, adhesion and invasion of ovarian cancer cells, while overexpression of E-cadherin decreased the invasion of ovarian cancer cells.

Effects of noise-induced hearing loss on parvalbumin and perineuronal net expression in the mouse primary auditory cortex.

Science.gov (United States)

Nguyen, Anna; Khaleel, Haroun M; Razak, Khaleel A

2017-07-01

Noise induced hearing loss is associated with increased excitability in the central auditory system but the cellular correlates of such changes remain to be characterized. Here we tested the hypothesis that noise-induced hearing loss causes deterioration of perineuronal nets (PNNs) in the auditory cortex of mice. PNNs are specialized extracellular matrix components that commonly enwrap cortical parvalbumin (PV) containing GABAergic interneurons. Compared to somatosensory and visual cortex, relatively less is known about PV/PNN expression patterns in the primary auditory cortex (A1). Whether changes to cortical PNNs follow acoustic trauma remains unclear. The first aim of this study was to characterize PV/PNN expression in A1 of adult mice. PNNs increase excitability of PV+ inhibitory neurons and confer protection to these neurons against oxidative stress. Decreased PV/PNN expression may therefore lead to a reduction in cortical inhibition. The second aim of this study was to examine PV/PNN expression in superficial (I-IV) and deep cortical layers (V-VI) following noise trauma. Exposing mice to loud noise caused an increase in hearing threshold that lasted at least 30 days. PV and PNN expression in A1 was analyzed at 1, 10 and 30 days following the exposure. No significant changes were observed in the density of PV+, PNN+, or PV/PNN co-localized cells following hearing loss. However, a significant layer- and cell type-specific decrease in PNN intensity was seen following hearing loss. Some changes were present even at 1 day following noise exposure. Attenuation of PNN may contribute to changes in excitability in cortex following noise trauma. The regulation of PNN may open up a temporal window for altered excitability in the adult brain that is then stabilized at a new and potentially pathological level such as in tinnitus. Copyright © 2017 Elsevier B.V. All rights reserved.

Del-1 overexpression potentiates lung cancer cell proliferation and invasion

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Lee, Seung-Hwan; Kim, Dong-Young; Jing, Feifeng; Kim, Hyesoon [Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Yun, Chae-Ok [Department of Bioengineering, College of Engineering, Hanyang University, Seoul (Korea, Republic of); Han, Deok-Jong [Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Choi, Eun Young, E-mail: choieun@ulsan.ac.kr [Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul (Korea, Republic of)

2015-12-04

Developmental endothelial locus-1 (Del-1) is an endogenous anti-inflammatory molecule that is highly expressed in the lung and the brain and limits leukocyte migration to these tissues. We previously reported that the expression of Del-1 is positively regulated by p53 in lung endothelial cells. Although several reports have implicated the altered expression of Del-1 gene in cancer patients, little is known about its role in tumor cells. We here investigated the effect of Del-1 on the features of human lung carcinoma cells. Del-1 mRNA was found to be significantly decreased in the human lung adenocarcinoma cell lines A549 (containing wild type of p53), H1299 (null for p53) and EKVX (mutant p53), compared to in human normal lung epithelial BEAS-2B cells and MRC-5 fibroblasts. The decrease of Del-1 expression was dependent on the p53 activity in the cell lines, but not on the expression of p53. Neither treatment with recombinant human Del-1 protein nor the introduction of adenovirus expressing Del-1 altered the expression of the apoptosis regulators BAX, PUMA and Bcl-2. Unexpectedly, the adenovirus-mediated overexpression of Del-1 gene into the lung carcinoma cell lines promoted proliferation and invasion of the lung carcinoma cells, as revealed by BrdU incorporation and transwell invasion assays, respectively. In addition, overexpression of the Del-1 gene enhanced features of epithelial–mesenchymal transition (EMT), such as increasing vimentin while decreasing E-cadherin in A549 cells, and increases in the level of Slug, an EMT-associated transcription regulator. Our findings demonstrated for the first time that there are deleterious effects of high levels of Del-1 in lung carcinoma cells, and suggest that Del-1 may be used as a diagnostic or prognostic marker for cancer progression, and as a novel therapeutic target for lung carcinoma. - Highlights: • Developmental Endothelial Locus-1 (Del-1) expression is downregulated in human lung cancer cells. �

Del-1 overexpression potentiates lung cancer cell proliferation and invasion

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Lee, Seung-Hwan; Kim, Dong-Young; Jing, Feifeng; Kim, Hyesoon; Yun, Chae-Ok; Han, Deok-Jong; Choi, Eun Young

2015-01-01

Developmental endothelial locus-1 (Del-1) is an endogenous anti-inflammatory molecule that is highly expressed in the lung and the brain and limits leukocyte migration to these tissues. We previously reported that the expression of Del-1 is positively regulated by p53 in lung endothelial cells. Although several reports have implicated the altered expression of Del-1 gene in cancer patients, little is known about its role in tumor cells. We here investigated the effect of Del-1 on the features of human lung carcinoma cells. Del-1 mRNA was found to be significantly decreased in the human lung adenocarcinoma cell lines A549 (containing wild type of p53), H1299 (null for p53) and EKVX (mutant p53), compared to in human normal lung epithelial BEAS-2B cells and MRC-5 fibroblasts. The decrease of Del-1 expression was dependent on the p53 activity in the cell lines, but not on the expression of p53. Neither treatment with recombinant human Del-1 protein nor the introduction of adenovirus expressing Del-1 altered the expression of the apoptosis regulators BAX, PUMA and Bcl-2. Unexpectedly, the adenovirus-mediated overexpression of Del-1 gene into the lung carcinoma cell lines promoted proliferation and invasion of the lung carcinoma cells, as revealed by BrdU incorporation and transwell invasion assays, respectively. In addition, overexpression of the Del-1 gene enhanced features of epithelial–mesenchymal transition (EMT), such as increasing vimentin while decreasing E-cadherin in A549 cells, and increases in the level of Slug, an EMT-associated transcription regulator. Our findings demonstrated for the first time that there are deleterious effects of high levels of Del-1 in lung carcinoma cells, and suggest that Del-1 may be used as a diagnostic or prognostic marker for cancer progression, and as a novel therapeutic target for lung carcinoma. - Highlights: • Developmental Endothelial Locus-1 (Del-1) expression is downregulated in human lung cancer cells. �

Effects of clusterin over-expression on metastatic progression and therapy in breast cancer

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Chatterjee Namita

2010-03-01

Full Text Available Abstract Background Clusterin is a secreted glycoprotein that is upregulated in a variety of cell lines in response to stress, and enhances cell survival. A second nuclear isoform of clusterin that is associated with cell death has also been identified. The aim of this study was to determine the role(s of the secretory isoform in breast tumor progression and metastasis. Methods To investigate the role of secretory clusterin in the biology of breast cancer tumor growth and resistance to therapy we have engineered an MCF-7 cell line (MCF-7CLU that over-expresses clusterin. We have measured the in vitro effects of clusterin over-expression on cell cycle, cell death, and sensitivity to TNFalpha and tamoxifen. Using an orthotopic model of breast cancer, we have also determined the effects of over-expression of clusterin on tumor growth and metastatic progression. Results In vitro, over-expression of secretory clusterin alters the cell cycle kinetics and decreases the rate of cell death, resulting in the enhancement of cell growth. Over-expression of secretory clusterin also blocks the TNFalpha-mediated induction of p21 and abrogates the cleavage of Bax to t-Bax, rendering the MCF-7CLU cells significantly more resistant to the cytokine than the parental cells. Orthotopic primary tumors derived from MCF-7CLU cells grow significantly more rapidly than tumors derived from parental MCF-7 cells and, unlike the parental cells, metastasize frequently to the lungs. Conclusions These data suggest that secretory clusterin, which is frequently up-regulated in breast cancers by common therapies, including anti-estrogens, may play a significant role in tumor growth, metastatic progression and subsequent drug resistance in surviving cells.

RNA-seq analysis of unintended effects in transgenic wheat overexpressing the transcription factor GmDREB1

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Qiyan Jiang

2017-06-01

Full Text Available The engineering of plants with enhanced tolerance to abiotic stresses typically involves complex multigene networks and may therefore have a greater potential to introduce unintended effects than the genetic modification for simple monogenic traits. For this reason, it is essential to study the unintended effects in transgenic plants engineered for stress tolerance. We selected drought- and salt-tolerant transgenic wheat overexpressing the transcription factor, GmDREB1, to investigate unintended pleiotropic effects using RNA-seq analysis. We compared the transcriptome alteration of transgenic plants with that of wild-type plants subjected to salt stress as a control. We found that GmDREB1 overexpression had a minimal impact on gene expression under normal conditions. GmDREB1 overexpression resulted in transcriptional reprogramming of the salt response, but many of the genes with differential expression are known to mitigate salt stress and contribute incrementally to the enhanced stress tolerance of transgenic wheat. GmDREB1 overexpression did not activate unintended gene networks with respect to gene expression in the roots of transgenic wheat. This work is important for establishing a method of detecting unintended effects of genetic engineering and the safety of such traits with the development of marketable transgenic crops in the near future.

Overexpression of Indian hedgehog partially rescues short stature homeobox 2-overexpression-associated congenital dysplasia of the temporomandibular joint in mice

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LI, XIHAI; LIANG, WENNA; YE, HONGZHI; WENG, XIAPING; LIU, FAYUAN; LIN, PINGDONG; LIU, XIANXIANG

2015-01-01

The role of short stature homeobox 2 (shox2) in the development and homeostasis of the temporomandibular joint (TMJ) has been well documented. Shox2 is known to be expressed in the progenitor cells and perichondrium of the developing condyle. A previous study by our group reported that overexpression of shox2 leads to congenital dysplasia of the TMJ via downregulation of the Indian hedgehog (Ihh) signaling pathway, which is essential for embryonic disc primordium formation and mandibular condylar growth. To determine whether overexpression of Ihh may rescue the overexpression of shox2 leading to congenital dysplasia of the TMJ, a mouse model in which Ihh and shox2 were overexpressed (Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice) was utilized to assess the consequences of this overexpression on TMJ development during post-natal life. The results showed that the developmental process and expression levels of runt-related transcription factor 2 and sex determining region Y-box 9 in the TMJ of the Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice were similar to those in wild-type mice. Overexpression of Ihh rescued shox2 overexpression-associated reduction of extracellular matrix components. However, overexpression of Ihh did not inhibit the shox2 overexpression-associated increase of matrix metalloproteinases (MMPs) MMP9, MMP13 and apoptosis in the TMJ. These combinatory cellular and molecular defects appeared to account for the observed congenital dysplasia of TMJ, suggesting that overexpression of Ihh partially rescued shox2 overexpression-associated congenital dysplasia of the TMJ in mice. PMID:26096903

Comparison of HER2 gene amplification and KRAS alteration in eyelid sebaceous carcinomas with that in other eyelid tumors.

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Kwon, Mi Jung; Shin, Hyung Sik; Nam, Eun Sook; Cho, Seong Jin; Lee, Min Joung; Lee, Samuel; Park, Hye-Rim

2015-05-01

Eyelid sebaceous carcinoma (SC) represents a highly aggressive malignancy. Despite the poor prognosis, genetic alterations as potential molecular targets are not available. KRAS mutation and HER2 gene amplification may be candidates related to their genetic alterations. We examined the HER2 and KRAS alteration status in eyelid SCs and compared it with that in other eyelid tumors. The controversial topics of the human papillomavirus (HPV) and p16 expression were also investigated. HER2 amplification was determined by silver in situ hybridization, while immunohistochemistry was performed to study protein expressions in 14 SCs and controls, including 23 other eyelid malignancies and 14 benign tumors. Peptide nucleic acid-mediated PCR clamping and direct sequencing were used to detect KRAS mutations. HER2 protein overexpression was observed in 85.7% (12/14) of the SCs, of which two-thirds showed HER2 gene amplification. HER2 protein overexpression and HER2 amplification were found more frequently in eyelid SCs than in other eyelid tumors. All SCs harbored wild type KRAS genes. No HPV infections were identified in the SCs. Nevertheless, p16 overexpression was found in 71.4% (10/14) of SCs, irrespective of the status of HPV infection. Furthermore, p16 overexpression in eyelid SCs was also significantly higher than that in other eyelid tumors. HER2 protein overexpression, HER2 gene amplifications, and wild type KRAS genes are common in eyelid SCs. HER2 gene amplification may represent potential therapeutic targets for the treatment of eyelid SCs. Copyright © 2014 Elsevier GmbH. All rights reserved.

Overexpression of neurofilament H disrupts normal cell structure and function

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Szebenyi, Gyorgyi; Smith, George M.; Li, Ping; Brady, Scott T.

2002-01-01

Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

Overexpression of Indian hedgehog partially rescues short stature homeobox 2-overexpression-associated congenital dysplasia of the temporomandibular joint in mice.

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Li, Xihai; Liang, Wenna; Ye, Hongzhi; Weng, Xiaping; Liu, Fayuan; Lin, Pingdong; Liu, Xianxiang

2015-09-01

The role of short stature homeobox 2 (shox2) in the development and homeostasis of the temporomandibular joint (TMJ) has been well documented. Shox2 is known to be expressed in the progenitor cells and perichondrium of the developing condyle. A previous study by our group reported that overexpression of shox2 leads to congenital dysplasia of the TMJ via downregulation of the Indian hedgehog (Ihh) signaling pathway, which is essential for embryonic disc primordium formation and mandibular condylar growth. To determine whether overexpression of Ihh may rescue the overexpression of shox2 leading to congenital dysplasia of the TMJ, a mouse model in which Ihh and shox2 were overexpressed (Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice) was utilized to assess the consequences of this overexpression on TMJ development during post-natal life. The results showed that the developmental process and expression levels of runt-related transcription factor 2 and sex determining region Y-box 9 in the TMJ of the Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice were similar to those in wild‑type mice. Overexpression of Ihh rescued shox2 overexpression-associated reduction of extracellular matrix components. However, overexpression of Ihh did not inhibit the shox2 overexpression-associated increase of matrix metalloproteinases (MMPs) MMP9, MMP13 and apoptosis in the TMJ. These combinatory cellular and molecular defects appeared to account for the observed congenital dysplasia of TMJ, suggesting that overexpression of Ihh partially rescued shox2 overexpression‑associated congenital dysplasia of the TMJ in mice.

Enhanced Stress Response in 5-HT1AR Overexpressing Mice: Altered HPA Function and Hippocampal Long-Term Potentiation.

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Pilar-Cuéllar, Fuencisla; Vidal, Rebeca; Díaz, Álvaro; Garro-Martínez, Emilio; Linge, Raquel; Castro, Elena; Haberzettl, Robert; Fink, Heidrun; Bert, Bettina; Brosda, Jan; Romero, Beatriz; Crespo-Facorro, Benedicto; Pazos, Ángel

2017-11-15

Postsynaptic 5-HT 1A receptors (5-HT 1A R) play an important role in anxiety and stress, although their contribution is still controversial. Previous studies report that mice overexpressing postsynaptic 5-HT 1A Rs show no changes in basal anxiety, though the influence of stress conditions has not been addressed yet. In this study, we used this animal model to evaluate the role of 5-HT 1A Rs in anxiety response after pre-exposure to an acute stressor. Under basal conditions, 5-HT 1A R overexpressing animals presented high corticosterone levels and a lower mineralocorticoid/glucocorticoid receptor ratio. After pre-exposure to a single stressor, they showed a high anxiety-like response, associated with a blunted increase in corticosterone levels and higher c-Fos activation in the prefrontal cortex. Moreover, these mice also presented a lack of downregulation of hippocampal long-term potentiation after stress exposure. Therefore, higher postsynaptic 5-HT 1A R activation might predispose to a high anxious phenotype and an impaired stress coping behavior.

The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

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Anita Collavoli

2011-01-01

Full Text Available By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB. This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

Prefrontal cortical parvalbumin and somatostatin expression and cell density increase during adolescence and are modified by BDNF and sex.

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Du, X; Serena, K; Hwang, W; Grech, A M; Wu, Y W C; Schroeder, A; Hill, R A

2018-04-01

Brain-derived neurotrophic factor (BDNF) is known to play a critical role early in the development of cortical GABAergic interneurons. Recently our laboratory and others have shown protracted development of specific subpopulations of GABAergic interneurons extending into adolescence. BDNF expression also changes significantly across adolescent development. However the role of BDNF in regulating GABAergic changes across adolescence remains unclear. Here, we performed a week-by-week analysis of the protein expression and cell density of three major GABAergic interneurons, parvalbumin (PV), somatostatin (SST) and calretinin (Cal) in the medial prefrontal cortex from prepubescence (week 3) to adulthood (week 12). In order to assess how BDNF and sex might influence the adolescent trajectory of GABAergic interneurons we compared WT as well as BDNF heterozygous (+/-) male and female mice. In both males and females PV expression increases during adolescent development in the mPFC. Compared to wild-types, PV expression was reduced in male but not female BDNF+/- mice throughout adolescent development. This reduction in protein expression corresponded with reduced cell density, specifically within the infralimbic prefrontal cortex. SST expression increased in early adolescent WT females and this upregulation was delayed in BDNF+/-. SST cell density also increased in early adolescent mPFC of WT female mice, with BDNF+/- again showing a reduced pattern of expression. Cal protein expression was also sex-dependently altered across adolescence with WT males showing a steady decline but that of BDNF+/- remaining unaltered. Reduced cell density in on the other hand was observed particularly in male BDNF+/- mice. In females, Cal protein expression and cell density remained largely stable. Our results show that PV, SST and calretinin interneurons are indeed still developing into early adolescence in the mPFC and that BDNF plays a critical, sex-specific role in mediating expression and

Prenatal acoustic stimulation influences neuronal size and the expression of calcium-binding proteins (calbindin D-28K and parvalbumin) in chick hippocampus.

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Chaudhury, Sraboni; Nag, Tapas Chandra; Wadhwa, Shashi

2006-12-01

Prenatal auditory enrichment by species-specific sounds and sitar music enhances the expression of immediate early genes, synaptic proteins and calcium binding proteins (CaBPs) as well as modifies the structural components of the brainstem auditory nuclei and auditory imprinting area in chicks. There is also facilitation of postnatal auditory preference of the chicks to maternal calls following both types of sound stimulation indicating prenatal perceptual learning. To examine whether the sound enrichment protocol also affects the areas related to learning and memory, we assessed morphological changes in the hippocampus at post-hatch day 1 of control and prenatally sound-stimulated chicks. Additionally, the proportions of neurons containing calbindin D-28K and parvalbumin immunoreactivity as well as their protein levels were determined. Fertilized eggs of domestic chick were incubated under normal conditions of temperature, humidity, forced draft of air as well as light and dark (12:12h) photoperiods. They were exposed to patterned sounds of species-specific and sitar music at 65 dB for 15 min per hour over a day/night cycle from day 10 of incubation till hatching. The hippocampal volume, neuronal nuclear size and total number of neurons showed a significant increase in the music-stimulated group as compared to the species-specific sound-stimulated and control groups. However, in both the auditory-stimulated groups the protein levels of calbindin and parvalbumin as well as the percentage of the immunopositive neurons were increased. The enhanced proportion of CaBPs in the sound-enriched groups suggests greater Ca(2+) influx, which may influence long-term potentiation and short-term memory.

C-MET overexpression and amplification in gliomas.

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Kwak, Yoonjin; Kim, Seong-Ik; Park, Chul-Kee; Paek, Sun Ha; Lee, Soon-Tae; Park, Sung-Hye

2015-01-01

We investigated c-Met overexpression and MET gene amplification in gliomas to determine their incidence and prognostic significance. c-Met immunohistochemistry and MET gene fluorescence in situ hybridization were carried out on tissue microarrays from 250 patients with gliomas (137 grade IV GBMs and 113 grade II and III diffuse gliomas). Clinicopathological features of these cases were reviewed. c-Met overexpression and MET gene amplification were detected in 13.1% and 5.1% of the GBMs, respectively. All the MET-amplified cases showed c-Met overexpression, but MET amplification was not always concordant with c-Met overexpression. None of grade II and III gliomas demonstrated c-Met overexpression or MET gene amplification. Mean survival of the GBM patients with MET amplification was not significantly different from patients without MET amplification (P=0.155). However, GBM patients with c-Met overexpression survived longer than patients without c-Met overexpression (P=0.035). Although MET amplification was not related to poor GBM prognosis, it is partially associated with the aggressiveness of gliomas, as MET amplification was found only in grade IV, not in grade II and III gliomas. We suggest that MET inhibitor therapy may be beneficial in about 5% GBMs, which was the incidence of MET gene amplification found in the patients included in this study.

Transcriptome response signatures associated with the overexpression of a mitochondrial uncoupling protein (AtUCP1 in tobacco.

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Alessandra Vasconcellos Nunes Laitz

Full Text Available Mitochondrial inner membrane uncoupling proteins (UCP dissipate the proton electrochemical gradient established by the respiratory chain, thus affecting the yield of ATP synthesis. UCP overexpression in plants has been correlated with oxidative stress tolerance, improved photosynthetic efficiency and increased mitochondrial biogenesis. This study reports the main transcriptomic responses associated with the overexpression of an UCP (AtUCP1 in tobacco seedlings. Compared to wild-type (WT, AtUCP1 transgenic seedlings showed unaltered ATP levels and higher accumulation of serine. By using RNA-sequencing, a total of 816 differentially expressed genes between the investigated overexpressor lines and the untransformed WT control were identified. Among them, 239 were up-regulated and 577 were down-regulated. As a general response to AtUCP1 overexpression, noticeable changes in the expression of genes involved in energy metabolism and redox homeostasis were detected. A substantial set of differentially expressed genes code for products targeted to the chloroplast and mainly involved in photosynthesis. The overall results demonstrate that the alterations in mitochondrial function provoked by AtUCP1 overexpression require important transcriptomic adjustments to maintain cell homeostasis. Moreover, the occurrence of an important cross-talk between chloroplast and mitochondria, which culminates in the transcriptional regulation of several genes involved in different pathways, was evidenced.

Altering textural properties of fermented milk by using surface-engineered Lactococcus lactis.

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Tarazanova, Mariya; Huppertz, Thom; Kok, Jan; Bachmann, Herwig

2018-05-09

Lactic acid bacteria are widely used for the fermentation of dairy products. While bacterial acidification rates, proteolytic activity and the production of exopolysaccharides are known to influence textural properties of fermented milk products, little is known about the role of the microbial surface on microbe-matrix interactions in dairy products. To investigate how alterations of the bacterial cell surface affect fermented milk properties, 25 isogenic Lactococcus lactis strains that differed with respect to surface charge, hydrophobicity, cell chaining, cell-clumping, attachment to milk proteins, pili expression and EPS production were used to produce fermented milk. We show that overexpression of pili increases surface hydrophobicity of various strains from 3-19% to 94-99%. A profound effect of different cell surface properties was an altered spatial distribution of the cells in the fermented product. Aggregated cells tightly fill the cavities of the protein matrix, while chaining cells seem to be localized randomly. A positive correlation was found between pili overexpression and viscosity and gel hardness of fermented milk. Gel hardness also positively correlated with clumping of cells in the fermented milk. Viscosity of fermented milk was also higher when it was produced with cells with a chaining phenotype or with cells that overexpress exopolysaccharides. Our results show that alteration of cell surface morphology affects textural parameters of fermented milk and cell localization in the product. This is indicative of a cell surface-dependent potential of bacterial cells as structure elements in fermented foods. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Cortical Gene Expression After a Conditional Knockout of 67 kDa Glutamic Acid Decarboxylase in Parvalbumin Neurons.

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Georgiev, Danko; Yoshihara, Toru; Kawabata, Rika; Matsubara, Takurou; Tsubomoto, Makoto; Minabe, Yoshio; Lewis, David A; Hashimoto, Takanori

2016-07-01

In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis. © The Author 2016. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

CEP55 overexpression predicts poor prognosis in patients with locally advanced esophageal squamous cell carcinoma.

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Jiang, Wenpeng; Wang, Zhou; Jia, Yang

2017-01-01

Development of esophageal squamous cell carcinoma (ESCC) involves alterations in multiple genes with corresponding proteins. Recent studies have demonstrated that centrosomal protein 55 (CEP55) shares certain features with oncogenes, and CEP55 overexpression is associated with the development and progression of malignant tumors. The present study aimed to analyze, for the first time, whether CEP55 expression is related to clinicopothalogic features in the esophageal squamous cell carcinoma (ESCC), as well as patient survival. A total of 110 patients with mid-thoracic ESCC who suffered from Ivor-Lewis were enrolled. The CEP55 expression profile of these patients in tumour tissues and corresponding healthy esophageal mucosa (CHEM) was detected by immunohistochemistry and semi-quantitative reverse transcription-polymerase chain reaction analyses. Correlations between CEP55 expression and clinicopathological factors were analyzed using χ 2 test. The log-rank test was employed to calculate survival rate. A Cox regression multivariate analysis was performed to determine independent prognostic factors. The results demonstrated that CEP55 expression in ESCC was significantly higher than that of CHEM (POverexpression of CEP55 was significantly associated with differentiation degree (P=0.022), T stage (P=0.019), lymph node metastasis (P=0.033), clinicopathological staging (P=0.002) and tumor recurrence (P=0.021) in locally advanced ESCC patients. In addition, CEP55 overexpression was significantly associated with reduced overall survival of patients after surgery (P=0.012). The 5-year survival rate of patients without CEP55 overexpression was significantly higher than that of patients with CEP55 overexpression (P=0.012). Therefore, these findings suggest that CEP55 overexpression correlates with poor prognosis in locally advanced ESCC patients.

Bmi1 overexpression in the cerebellar granule cell lineage of mice affects cell proliferation and survival without initiating medulloblastoma formation

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Hourinaz Behesti

2013-01-01

BMI1 is a potent inducer of neural stem cell self-renewal and neural progenitor cell proliferation during development and in adult tissue homeostasis. It is overexpressed in numerous human cancers – including medulloblastomas, in which its functional role is unclear. We generated transgenic mouse lines with targeted overexpression of Bmi1 in the cerebellar granule cell lineage, a cell type that has been shown to act as a cell of origin for medulloblastomas. Overexpression of Bmi1 in granule cell progenitors (GCPs led to a decrease in cerebellar size due to decreased GCP proliferation and repression of the expression of cyclin genes, whereas Bmi1 overexpression in postmitotic granule cells improved cell survival in response to stress by altering the expression of genes in the mitochondrial cell death pathway and of Myc and Lef-1. Although no medulloblastomas developed in ageing cohorts of transgenic mice, crosses with Trp53−/− mice resulted in a low incidence of medulloblastoma formation. Furthermore, analysis of a large collection of primary human medulloblastomas revealed that tumours with a BMI1high TP53low molecular profile are significantly enriched in Group 4 human medulloblastomas. Our data suggest that different levels and timing of Bmi1 overexpression yield distinct cellular outcomes within the same cellular lineage. Importantly, Bmi1 overexpression at the GCP stage does not induce tumour formation, suggesting that BMI1 overexpression in GCP-derived human medulloblastomas probably occurs during later stages of oncogenesis and might serve to enhance tumour cell survival.

Perturbation of Auxin Homeostasis and Signaling by PINOID Overexpression Induces Stress Responses in Arabidopsis

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Kumud Saini

2017-08-01

Full Text Available Under normal and stress conditions plant growth require a complex interplay between phytohormones and reactive oxygen species (ROS. However, details of the nature of this crosstalk remain elusive. Here, we demonstrate that PINOID (PID, a serine threonine kinase of the AGC kinase family, perturbs auxin homeostasis, which in turn modulates rosette growth and induces stress responses in Arabidopsis plants. Arabidopsis mutants and transgenic plants with altered PID expression were used to study the effect on auxin levels and stress-related responses. In the leaves of plants with ectopic PID expression an accumulation of auxin, oxidative burst and disruption of hormonal balance was apparent. Furthermore, PID overexpression led to the accumulation of antioxidant metabolites, while pid knockout mutants showed only moderate changes in stress-related metabolites. These physiological changes in the plants overexpressing PID modulated their response toward external drought and osmotic stress treatments when compared to the wild type. Based on the morphological, transcriptome, and metabolite results, we propose that perturbations in the auxin hormone levels caused by PID overexpression, along with other hormones and ROS downstream, cause antioxidant accumulation and modify growth and stress responses in Arabidopsis. Our data provide further proof for a strong correlation between auxin and stress biology.

Revolutionizing membrane protein overexpression in bacteria

NARCIS (Netherlands)

Schlegel, Susan; Klepsch, Mirjam; Gialama, Dimitra; Wickstrom, David; Slotboom, Dirk Jan; de Gier, Jan-Willem; Wickström, David

The bacterium Escherichia coli is the most widely used expression host for overexpression trials of membrane proteins. Usually, different strains, culture conditions and expression regimes are screened for to identify the optimal overexpression strategy. However, yields are often not satisfactory,

Tet1 overexpression leads to anxiety-like behavior and enhanced fear memories via the activation of calcium-dependent cascade through Egr1 expression in mice.

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Kwon, Wookbong; Kim, Hyeng-Soo; Jeong, Jain; Sung, Yonghun; Choi, Minjee; Park, Song; Lee, Jinhee; Jang, Soyoung; Kim, Sung Hyun; Lee, Sanggyu; Kim, Myoung Ok; Ryoo, Zae Young

2018-01-01

Ten-eleven translocation methylcytosine dioxygenase 1 ( Tet1 ) initiates DNA demethylation by converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) at CpG-rich regions of genes, which have key roles in adult neurogenesis and memory. In addition, the overexpression of Tet1 with 5-hmC alteration in patients with psychosis has also been reported, for instance in schizophrenia and bipolar disorders. The mechanism underlying Tet1 overexpression in the brain; however, is still elusive. In the present study, we found that Tet1-transgenic (Tet1-TG) mice displayed abnormal behaviors involving elevated anxiety and enhanced fear memories. We confirmed that Tet1 overexpression affected adult neurogenesis with oligodendrocyte differentiation in the hippocampal dentate gyrus of Tet1-TG mice. In addition, Tet1 overexpression induced the elevated expression of immediate early genes, such as Egr1 , c-fos , Arc , and Bdnf , followed by the activation of intracellular calcium signals ( i.e. , CamKII, ERK, and CREB) in prefrontal and hippocampal neurons. The expression of GABA receptor subunits ( Gabra2 and Gabra4 ) fluctuated in the prefrontal cortex and hippocampus. We evaluated the effects of Tet1 overexpression on intracellular calcium-dependent cascades by activating the Egr1 promoter in vitro Tet1 enhanced Egr1 expression, which may have led to alterations in Gabra2 and Gabra4 expression in neurons. Taken together, we suggest that the Tet1 overexpression in our Tet1-TG mice can be applied as an effective model for studying various stress-related diseases that show hyperactivation of intracellular calcium-dependent cascades in the brain.-Kwon, W., Kim, H.-S., Jeong, J., Sung, Y., Choi, M., Park, S., Lee, J., Jang, S., Kim, S. H., Lee, S., Kim, M. O., Ryoo, Z. Y. Tet1 overexpression leads to anxiety-like behavior and enhanced fear memories via the activation of calcium-dependent cascade through Egr1 expression in mice. © FASEB.

Clinical significance of altered nm23-H1, EGFR, RB and p53 expression in bilharzial bladder cancer

International Nuclear Information System (INIS)

Khaled, Hussein M; Bahnassy, Abeer A; Raafat, Amira A; Zekri, Abdel-Rahman N; Madboul, Maha S; Mokhtar, Nadia M

2009-01-01

Clinical characterization of bladder carcinomas is still inadequate using the standard clinico-pathological prognostic markers. We assessed the correlation between nm23-H1, Rb, EGFR and p53 in relation to the clinical outcome of patients with muscle invasive bilharzial bladder cancer (MI-BBC). nm23-H1, Rb, EGFR and p53 expression was assessed in 59 MI-BBC patients using immunohistochemistry and reverse transcription (RT-PCR) and was correlated to the standard clinico-pathological prognostic factors, patient's outcome and the overall survival (OS) rate. Overexpression of EGFR and p53 proteins was detected in 66.1% and 35.6%; respectively. Loss of nm23-H1and Rb proteins was detected in 42.4% and 57.6%; respectively. Increased EGFR and loss of nm23-H1 RNA were detected in 61.5% and 36.5%; respectively. There was a statistically significant correlation between p53 and EGFR overexpression (p < 0.0001), nm23 loss (protein and RNA), lymph node status (p < 0.0001); between the incidence of local recurrence and EGFR RNA overexpression (p= 0.003) as well as between the incidence of metastasis and altered Rb expression (p = 0.026), p53 overexpression (p < 0.0001) and mutation (p = 0.04). Advanced disease stage correlated significantly with increased EGFR (protein and RNA) (p = 0.003 & 0.01), reduced nm23-H1 RNA (p = 0.02), altered Rb (p = 0.023), and p53 overexpression (p = 0.004). OS rates correlated significantly, in univariate analysis, with p53 overexpression (p = 0.011), increased EGFR (protein and RNA, p = 0.034&0.031), nm23-H1 RNA loss (p = 0.021) and aberrations of ≥ 2 genes. However, multivariate analysis showed that only high EGFR overexpression, metastatic recurrence, high tumor grade and the combination of ≥ 2 affected markers were independent prognostic factors. nm23-H1, EGFR and p53 could be used as prognostic biomarkers in MI-BBC patients. In addition to the standard pathological prognostic factors, a combination of these markers (≥ 2) has

Core I gene is overexpressed in Hürthle and non-Hürthle cell microfollicular adenomas and follicular carcinomas of the thyroid

International Nuclear Information System (INIS)

Máximo, Valdemar; Preto, Ana; Crespo, Ana; Rocha, Ana Sofia; Machado, José Carlos; Soares, Paula; Sobrinho-Simões, Manuel

2004-01-01

Most of the steps involved in the initiation and progression of Hürthle (oncocytic, oxyphilic) cell carcinomas of the thyroid remain unknown. Using differential display and semiquantitative RT-PCR we found, among other alterations, overexpression of the gene encoding the Core I subunit of the complex III of the mitochondrial respiratory chain in a follicular carcinoma composed of Hürthle cells. Similar high levels of Core I gene expression were detected in nine follicular carcinomas (seven with Hürthle cell features), in seven microfollicular adenomas (one with Hürthle cell features) and in one micro/macrofollicular adenoma, in contrast to a lower/normal expression in nine papillary carcinomas (three with Hürthle cell features) and five macrofollicular adenomas (one of which displaying Hürthle cell features). No significative correlation was found between Core I overexpression and the proliferative activity of the lesions. We conclude that Core I overexpression in thyroid tumours is not associated with malignancy, Hürthle cells or proliferative activity. The pathogenetic mechanism linking Core I overexpression to the microfollicular pattern of growth of thyroid tumours remains to be clarified

Homeobox B9 is overexpressed in hepatocellular carcinomas and promotes tumor cell proliferation both in vitro and in vivo

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Li, Fangyi; Dong, Lei; Xing, Rong; Wang, Li; Luan, Fengming; Yao, Chenhui; Ji, Xuening; Bai, Lizhi

2014-01-01

Highlights: • HOXB9 is overexpressed in human HCC samples. • HOXB9 over expression had shorter survival time than down expression. • HOXB9 stimulated the proliferation of HCC cells. • Activation of TGF-β1 contributes to HOXB9-induced proliferation in HCC cells. - Abstract: HomeoboxB9 (HOXB9), a nontransforming transcription factor that is overexpressed in multiple tumor types, alters tumor cell fate and promotes tumor progression. However, the role of HOXB9 in hepatocellular carcinoma (HCC) development has not been well studied. In this paper, we found that HOXB9 is overexpressed in human HCC samples. We investigated HOXB9 expression and its prognostic value for HCC. HCC surgical tissue samples were taken from 89 HCC patients. HOXB9 overexpression was observed in 65.2% of the cases, and the survival analysis showed that the HOXB9 overexpression group had significantly shorter overall survival time than the HOXB9 downexpression group. The ectopic expression of HOXB9 stimulated the proliferation of HCC cells; whereas the knockdown of HOXB9 produced an opposite effect. HOXB9 also modulated the tumorigenicity of HCC cells in vivo. Moreover, we found that the activation of TGF-β1 contributes to HOXB9-induced proliferation activities. The results provide the first evidence that HOXB9 is a critical regulator of tumor growth factor in HCC

Homeobox B9 is overexpressed in hepatocellular carcinomas and promotes tumor cell proliferation both in vitro and in vivo

Energy Technology Data Exchange (ETDEWEB)

Li, Fangyi [Department of General Surgery, Dalian Municipal Friendship Hospital, No. 8 Sanba Square, Zhongshan District, Dalian 116001 (China); Dong, Lei, E-mail: dlleidong@126.com [Department of Laparoscopic Surgery, First Affiliated Hospital of Dalian Medical University, No. 193 Lianhe Street, Shahekou District, Dalian 116001 (China); Xing, Rong [Department of Pathology and Pathophysiology, Dalian Medical University, No. 9 Lvshunnan Road, Lvshunkou District, Dalian 116044 (China); Wang, Li; Luan, Fengming; Yao, Chenhui [Department of General Surgery, Dalian Municipal Friendship Hospital, No. 8 Sanba Square, Zhongshan District, Dalian 116001 (China); Ji, Xuening [Department of Oncology, Zhongshan Hospital of Dalian University, No. 6 Jiefang Street, Zhongshan District, Dalian 116001 (China); Bai, Lizhi, E-mail: dllizhibai@126.com [Department of Emergency, Zhongshan Hospital of Dalian University, No. 6 Jiefang Street, Zhongshan District, Dalian 116001 (China)

2014-02-07

Highlights: • HOXB9 is overexpressed in human HCC samples. • HOXB9 over expression had shorter survival time than down expression. • HOXB9 stimulated the proliferation of HCC cells. • Activation of TGF-β1 contributes to HOXB9-induced proliferation in HCC cells. - Abstract: HomeoboxB9 (HOXB9), a nontransforming transcription factor that is overexpressed in multiple tumor types, alters tumor cell fate and promotes tumor progression. However, the role of HOXB9 in hepatocellular carcinoma (HCC) development has not been well studied. In this paper, we found that HOXB9 is overexpressed in human HCC samples. We investigated HOXB9 expression and its prognostic value for HCC. HCC surgical tissue samples were taken from 89 HCC patients. HOXB9 overexpression was observed in 65.2% of the cases, and the survival analysis showed that the HOXB9 overexpression group had significantly shorter overall survival time than the HOXB9 downexpression group. The ectopic expression of HOXB9 stimulated the proliferation of HCC cells; whereas the knockdown of HOXB9 produced an opposite effect. HOXB9 also modulated the tumorigenicity of HCC cells in vivo. Moreover, we found that the activation of TGF-β1 contributes to HOXB9-induced proliferation activities. The results provide the first evidence that HOXB9 is a critical regulator of tumor growth factor in HCC.

[Epigenetic alterations in acute lymphoblastic leukemia].

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Navarrete-Meneses, María Del Pilar; Pérez-Vera, Patricia

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. It is well-known that genetic alterations constitute the basis for the etiology of ALL. However, genetic abnormalities are not enough for the complete development of the disease, and additional alterations such as epigenetic modifications are required. Such alterations, like DNA methylation, histone modifications, and noncoding RNA regulation have been identified in ALL. DNA hypermethylation in promoter regions is one of the most frequent epigenetic modifications observed in ALL. This modification frequently leads to gene silencing in tumor suppressor genes, and in consequence, contributes to leukemogenesis. Alterations in histone remodeling proteins have also been detected in ALL, such as the overexpression of histone deacetylases enzymes, and alteration of acetyltransferases and methyltransferases. ALL also shows alteration in the expression of miRNAs, and in consequence, the modification in the expression of their target genes. All of these epigenetic modifications are key events in the malignant transformation since they lead to the deregulation of oncogenes as BLK, WNT5B and WISP1, and tumor suppressors such as FHIT, CDKN2A, CDKN2B, and TP53, which alter fundamental cellular processes and potentially lead to the development of ALL. Both genetic and epigenetic alterations contribute to the development and evolution of ALL. Copyright © 2017 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.

Convergent differential regulation of parvalbumin in the brains of vocal learners.

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Erina Hara

Full Text Available Spoken language and learned song are complex communication behaviors found in only a few species, including humans and three groups of distantly related birds--songbirds, parrots, and hummingbirds. Despite their large phylogenetic distances, these vocal learners show convergent behaviors and associated brain pathways for vocal communication. However, it is not clear whether this behavioral and anatomical convergence is associated with molecular convergence. Here we used oligo microarrays to screen for genes differentially regulated in brain nuclei necessary for producing learned vocalizations relative to adjacent brain areas that control other behaviors in avian vocal learners versus vocal non-learners. A top candidate gene in our screen was a calcium-binding protein, parvalbumin (PV. In situ hybridization verification revealed that PV was expressed significantly higher throughout the song motor pathway, including brainstem vocal motor neurons relative to the surrounding brain regions of all distantly related avian vocal learners. This differential expression was specific to PV and vocal learners, as it was not found in avian vocal non-learners nor for control genes in learners and non-learners. Similar to the vocal learning birds, higher PV up-regulation was found in the brainstem tongue motor neurons used for speech production in humans relative to a non-human primate, macaques. These results suggest repeated convergent evolution of differential PV up-regulation in the brains of vocal learners separated by more than 65-300 million years from a common ancestor and that the specialized behaviors of learned song and speech may require extra calcium buffering and signaling.

The HGF Receptor c-Met Is Overexpressed in Esophageal Adenocarcinoma

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Luis J. Herrera

2005-01-01

Full Text Available The hepatocyte growth factor (HGF receptor, Met, has established oncogenic properties; however, its expression and function in esophageal adenocarcinoma (EA remain poorly understood. We aimed to determine the expression and potential alterations in Met expression in EA. Met expression was investigated in surgical specimens of EA, Barrett's esophagus (BE, and normal esophagus (NE using immunohistochemistry (IHC and quantitative reverse transcriptase polymerase chain reaction. Met expression, phosphorylation, and the effect of COX-2 inhibition on expression were examined in EA cell lines. IHC demonstrated intense Met immunoreactivity in all (100% EA and dysplastic BE specimens. In contrast, minimal immunostaining was observed in BE without dysplasia or NE specimens. Met mRNA and protein levels were increased in three EA cell lines, and Met protein was phosphorylated in the absence of serum. Sequence analysis found the kinase domain of c-met to be wild type in all three EA cell lines. HGF mRNA expression was identified in two EA cell lines. In COX-2-overexpressing cells, COX-2 inhibition decreased Met expression. Met is consistently overexpressed in EA surgical specimens and in three EA cell lines. Met dysregulation occurs early in Barrett's dysplasia to adenocarcinoma sequence. Future study of Met inhibition as a potential biologic therapy for EA is warranted.

SNEV overexpression extends the life span of human endothelial cells

International Nuclear Information System (INIS)

Voglauer, Regina; Chang, Martina Wei-Fen; Dampier, Brigitta; Wieser, Matthias; Baumann, Kristin; Sterovsky, Thomas; Schreiber, Martin; Katinger, Hermann; Grillari, Johannes

2006-01-01

In a recent screening for genes downregulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients. Since these mRNA profiles suggested a role of SNEV in the regulation of cell proliferation, the effect of its overexpression was tested. Thereby, a significant extension of the cellular life span was observed, which was not caused by altered telomerase activity or telomere dynamics but rather by enhanced stress resistance. When SNEV overexpressing cells were treated with bleomycin or bleomycin combined with BSO, inducing DNA damage as well as reactive oxygen species, a significantly lower fraction of apoptotic cells was found in comparison to vector control cells. These data suggest that high levels of SNEV might extend the cellular life span by increasing the resistance to stress or by improving the DNA repair capacity of the cells

Structural and Functional Alterations in Neocortical Circuits after Mild Traumatic Brain Injury

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Vascak, Michal

National concern over traumatic brain injury (TBI) is growing rapidly. Recent focus is on mild TBI (mTBI), which is the most prevalent injury level in both civilian and military demographics. A preeminent sequelae of mTBI is cognitive network disruption. Advanced neuroimaging of mTBI victims supports this premise, revealing alterations in activation and structure-function of excitatory and inhibitory neuronal systems, which are essential for network processing. However, clinical neuroimaging cannot resolve the cellular and molecular substrates underlying such changes. Therefore, to understand the full scope of mTBI-induced alterations it is necessary to study cortical networks on the microscopic level, where neurons form local networks that are the fundamental computational modules supporting cognition. Recently, in a well-controlled animal model of mTBI, we demonstrated in the excitatory pyramidal neuron system, isolated diffuse axonal injury (DAI), in concert with electrophysiological abnormalities in nearby intact (non-DAI) neurons. These findings were consistent with altered axon initial segment (AIS) intrinsic activity functionally associated with structural plasticity, and/or disturbances in extrinsic systems related to parvalbumin (PV)-expressing interneurons that form GABAergic synapses along the pyramidal neuron perisomatic/AIS domains. The AIS and perisomatic GABAergic synapses are domains critical for regulating neuronal activity and E-I balance. In this dissertation, we focus on the neocortical excitatory pyramidal neuron/inhibitory PV+ interneuron local network following mTBI. Our central hypothesis is that mTBI disrupts neuronal network structure and function causing imbalance of excitatory and inhibitory systems. To address this hypothesis we exploited transgenic and cre/lox mouse models of mTBI, employing approaches that couple state-of-the-art bioimaging with electrophysiology to determine the structuralfunctional alterations of excitatory and

Gastrointestinal hyperplasia with altered expression of DNA polymerase beta.

Directory of Open Access Journals (Sweden)

Katsuhiko Yoshizawa

2009-08-01

Full Text Available Altered expression of DNA polymerase beta (Pol beta has been documented in a large percentage of human tumors. However, tumor prevalence or predisposition resulting from Pol beta over-expression has not yet been evaluated in a mouse model.We have recently developed a novel transgenic mouse model that over-expresses Pol beta. These mice present with an elevated incidence of spontaneous histologic lesions, including cataracts, hyperplasia of Brunner's gland and mucosal hyperplasia in the duodenum. In addition, osteogenic tumors in mice tails, such as osteoma and osteosarcoma were detected. This is the first report of elevated tumor incidence in a mouse model of Pol beta over-expression. These findings prompted an evaluation of human gastrointestinal tumors with regard to Pol beta expression. We observed elevated expression of Pol beta in stomach adenomas and thyroid follicular carcinomas, but reduced Pol beta expression in esophageal adenocarcinomas and squamous carcinomas.These data support the hypothesis that balanced and proficient base excision repair protein expression and base excision repair capacity is required for genome stability and protection from hyperplasia and tumor formation.

Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional nonlinear feedback.

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Wilson, K; Auer, M; Binnie, M; Zheng, X; Pham, N T; Iredale, J P; Webster, S P; Mole, D J

2016-04-14

Kynurenine 3-monooxygenase (KMO) is a critical regulator of inflammation. The preferred KMO substrate, kynurenine, is converted to 3-hydroxykynurenine (3HK), and this product exhibits cytotoxicity through mechanisms that culminate in apoptosis. Here, we report that overexpression of human KMO with orthotopic localisation to mitochondria creates a metabolic environment during which the cell exhibits increased tolerance for exogenous 3HK-mediated cellular injury. Using the selective KMO inhibitor Ro61-8048, we show that KMO enzyme function is essential for cellular protection. Pan-caspase inhibition with Z-VAD-FMK confirmed apoptosis as the mode of cell death. By defining expression of pathway components upstream and downstream of KMO, we observed alterations in other key kynurenine pathway components, particularly tryptophan-2,3-dioxygenase upregulation, through bidirectional nonlinear feedback. KMO overexpression also increased expression of inducible nitric oxide synthase (iNOS). These changes in gene expression are functionally relevant, because siRNA knockdown of the pathway components kynureninase and quinolinate phosphoribosyl transferase caused cells to revert to a state of susceptibility to 3HK-mediated apoptosis. In summary, KMO overexpression, and importantly KMO activity, have metabolic repercussions that fundamentally affect resistance to cell stress.

Genomic Instability Promoted by Overexpression of Mismatch Repair Factors in Yeast: A Model for Understanding Cancer Progression.

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Chakraborty, Ujani; Dinh, Timothy A; Alani, Eric

2018-04-13

Mismatch repair (MMR) proteins act in spellchecker roles to excise misincorporation errors that occur during DNA replication. Curiously, large-scale analyses of a variety of cancers showed that increased expression of MMR proteins often correlated with tumor aggressiveness, metastasis, and early recurrence. To better understand these observations, we used the TCGA and GENT databases to analyze MMR protein expression in cancers. We found that the MMR genes MSH2 and MSH6 are overexpressed more frequently than MSH3 , and that MSH2 and MSH6 are often co-overexpressed as a result of copy number amplifications of these genes. These observations encouraged us to test the effects of upregulating MMR protein levels in baker's yeast, where we can sensitively monitor genome instability phenotypes associated with cancer initiation and progression. Msh6 overexpression (2 to 4-fold) almost completely disrupted mechanisms that prevent recombination between divergent DNA sequences by interacting with the DNA polymerase processivity clamp PCNA and by sequestering the Sgs1 helicase. Importantly, co-overexpression of Msh2 and Msh6 (∼8-fold) conferred, in a PCNA interaction dependent manner, several genome instability phenotypes including increased mutation rate, increased sensitivity to the DNA replication inhibitor hydroxyurea and the DNA damaging agents methyl methanesulfonate and 4-nitroquinoline N-oxide, and elevated loss of heterozygosity. Msh2 and Msh6 co-overexpression also altered the cell cycle distribution of exponentially growing cells, resulting in an increased fraction of unbudded cells, consistent with a larger percentage of cells in G1. These novel observations suggested that overexpression of MSH factors affected the integrity of the DNA replication fork, causing genome instability phenotypes that could be important for promoting cancer progression. Copyright © 2018, Genetics.

Calmodulin overexpression does not alter Cav1.2 function or oligomerization state.

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Findeisen, Felix; Tolia, Alexandra; Arant, Ryan; Kim, Eun Young; Isacoff, Ehud; Minor, Daniel L

2011-01-01

Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca(v)s) are crucial for Ca(v) activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca(2+)/CaM molecules bound to the Ca(v)1.2 C terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca(2+)/CaM bridged two PreIQ helixes to form a 4:2 Ca(2+)/CaM:Ca(v) C-terminal tail assembly. Contrary to previous proposals based on a similar crystallographic dimer, extensive biochemical analysis together with subunit counting experiments of full-length channels in live cell membranes failed to find evidence for multimers that would be compatible with the 4:2 crossbridged complex. Here, we examine this possibility further. We find that CaM over-expression has no functional effect on Ca(v)1.2 inactivation or on the stoichiometry of full-length Ca(v)1.2. These data provide further support for the monomeric Ca(v)1.2 stoichiometry. Analysis of the electrostatic surfaces of the 2:1 Ca(2+)/CaM:Ca(V) C-terminal tail assembly reveals notable patches of electronegativity. These could influence various forms of channel modulation by interacting with positively charged elements from other intracellular channel domains.

Neuroglobin Overexpression Inhibits AMPK Signaling and Promotes Cell Anabolism.

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Cai, Bin; Li, Wenjun; Mao, XiaoOu; Winters, Ali; Ryou, Myoung-Gwi; Liu, Ran; Greenberg, David A; Wang, Ning; Jin, Kunlin; Yang, Shao-Hua

2016-03-01

Neuroglobin (Ngb) is a recently discovered globin with preferential localization to neurons. Growing evidence indicates that Ngb has distinct physiological functions separate from the oxygen storage and transport roles of other globins, such as hemoglobin and myoglobin. We found increased ATP production and decreased glycolysis in Ngb-overexpressing immortalized murine hippocampal cell line (HT-22), in parallel with inhibition of AMP-activated protein kinase (AMPK) signaling and activation of acetyl-CoA carboxylase (ACC). In addition, lipid and glycogen content was increased in Ngb-overexpressing HT-22 cells. AMPK signaling was also inhibited in the brain and heart from Ngb-overexpressing transgenic mice. Although Ngb overexpression did not change glycogen content in whole brain, glycogen synthase was activated in cortical neurons of Ngb-overexpressing mouse brain and Ngb overexpression primary neurons. Moreover, lipid and glycogen content was increased in hearts derived from Ngb-overexpressing mice. These findings suggest that Ngb functions as a metabolic regulator and enhances cellular anabolism through the inhibition of AMPK signaling.

Overexpression of Catalase in Vascular Smooth Muscle Cells Prevents the Formation of Abdominal Aortic Aneurysms

Science.gov (United States)

Parastatidis, Ioannis; Weiss, Daiana; Joseph, Giji; Taylor, W Robert

2013-01-01

Objective Elevated levels of oxidative stress have been reported in abdominal aortic aneurysms (AAA), but which reactive oxygen species (ROS) promotes the development of AAA remains unclear. Here we investigate the effect of the hydrogen peroxide (H2O2) degrading enzyme catalase on the formation of AAA. Approach and Results AAA were induced with the application of calcium chloride (CaCl2) on mouse infrarenal aortas. The administration of PEG-catalase, but not saline, attenuated the loss of tunica media and protected against AAA formation (0.91±0.1 mm vs. 0.76±0.09 mm). Similarly, in a transgenic mouse model, catalase over-expression in the vascular smooth muscle cells (VSMC) preserved the thickness of tunica media and inhibited aortic dilatation by 50% (0.85±0.14 mm vs. 0.57±0.08 mm). Further studies showed that injury with CaCl2 decreased catalase expression and activity in the aortic wall. Pharmacologic administration or genetic over-expression of catalase restored catalase activity and subsequently decreased matrix metalloproteinase activity. In addition, a profound reduction in inflammatory markers and VSMC apoptosis was evident in aortas of catalase over-expressing mice. Interestingly, as opposed to infusion of PEG-catalase, chronic over-expression of catalase in VSMC did not alter the total aortic H2O2 levels. Conclusions The data suggest that a reduction in aortic wall catalase activity can predispose to AAA formation. Restoration of catalase activity in the vascular wall enhances aortic VSMC survival and prevents AAA formation primarily through modulation of matrix metalloproteinase activity. PMID:23950141

Overexpression of hepatoma-derived growth factor in melanocytes does not lead to oncogenic transformation

International Nuclear Information System (INIS)

Sedlmaier, Angela; Wernert, Nicolas; Gallitzendörfer, Rainer; Abouzied, Mekky M; Gieselmann, Volkmar; Franken, Sebastian

2011-01-01

HDGF is a growth factor which is overexpressed in a wide range of tumors. Importantly, expression levels were identified as a prognostic marker in some types of cancer such as melanoma. To investigate the presumed oncogenic/transforming capacity of HDGF, we generated transgenic mice overexpressing HDGF in melanocytes. These mice were bred with mice heterozygous for a defective copy of the Ink4a tumor suppressor gene and were exposed to UV light to increase the risk for tumor development both genetically and physiochemically. Mice were analyzed by immunohistochemistry and Western blotting. Furthermore, primary melanocytes were isolated from different strains created. Transgenic animals overexpressed HDGF in hair follicle melanocytes. Interestingly, primary melanocytes isolated from transgenic animals were not able to differentiate in vitro whereas cells isolated from wild type and HDGF-deficient animals were. Although, HDGF -/- /Ink4a +/- mice displayed an increased number of epidermoid cysts after exposure to UV light, no melanomas or premelanocytic alterations could be detected in this mouse model. The results therefore provide no evidence that HDGF has a transforming capacity in tumor development. Our results in combination with previous findings point to a possible role in cell differentiation and suggest that HDGF promotes tumor progression after secondary upregulation and may represent another protein fitting into the concept of non-oncogene addiction of tumor tissue

Mammary gland tumor formation in transgenic mice overexpressing stromelysin-1

Energy Technology Data Exchange (ETDEWEB)

Sympson, Carolyn J; Bissell, Mina J; Werb, Zena

1995-06-01

An intact basement membrane (BM) is essential for the proper function, differentiation and morphology of many epithelial cells. The disruption or loss of this BM occurs during normal development as well as in the disease state. To examine the importance of BM during mammary gland development in vivo, we generated transgenic mice that inappropriately express autoactivating isoforms of the matrix metalloproteinase stromelysin-1. The mammary glands from these mice are both functionally and morphologically altered throughout development. We have now documented a dramatic incidence of breast tumors in several independent lines of these mice. These data suggest that overexpression of stromelysin-1 and disruption of the BM may be a key step in the multi-step process of breast cancer.

β‑catenin nuclear translocation induced by HIF‑1α overexpression leads to the radioresistance of prostate cancer.

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Luo, Yong; Li, Mingchuan; Zuo, Xuemei; Basourakos, Spyridon P; Zhang, Jiao; Zhao, Jiahui; Han, Yili; Lin, Yunhua; Wang, Yongxing; Jiang, Yongguang; Lan, Ling

2018-04-12

Hypoxia-inducible factor‑1α (HIF‑1α) is known to play crucial roles in tumor radioresistance; however, the molecular mechanisms responsible for the promotion of tumor radioresistance by HIF‑1α remain unclear. β‑catenin is known to be involved in the metastatic potential of prostate cancer (PCa). In this study, to investigate the role of HIF‑1α and β‑catenin in the radioresistance of PCa, two PCa cell lines, LNCaP and C4‑2B, were grouped as follows: Negative control (no treatment), HIF‑1α overexpression group (transfected with HIF‑1α overexpression plasmid) and β‑catenin silenced group (transfected with HIF‑1α plasmids and β‑catenin-shRNA). Cell proliferation, cell cycle, cell invasion and radiosensitivity were examined under normal or hypoxic conditions. In addition, radiosensitivity was examined in two mouse PCa models (the LNCaP orthotopic BALB/c-nu mice model and the C4‑2B subcutaneous SCID mice model). Our results revealed that in both the LNCaP and C4‑2B cells, transfection with HIF‑1α overexpression plasmid led to an enhanced β‑catenin nuclear translocation, while β‑catenin silencing inhibited β‑catenin nuclear translocation. The enhanced β‑catenin nuclear translocation induced by HIF‑1α overexpression resulted in an enhanced cell proliferation and cell invasion, an altered cell cycle distribution, decreased apoptosis, and improved non‑homologous end joining (NHEJ) repair under normal and irradiation conditions. Similar results were observed in the animal models. HIF‑1α overexpression enhanced β‑catenin nuclear translocation, which led to the activation of the β‑catenin/NHEJ signaling pathway and increased cell proliferation, cell invasion and DNA repair. These results thus suggest that HIF‑1α overexpression promotes the radioresistance of PCa cells.

Glycomic and sialoproteomic data of gastric carcinoma cells overexpressing ST3GAL4

DEFF Research Database (Denmark)

Mereiter, Stefan; Magalhães, Ana; Adamczyk, Barbara

2016-01-01

Gastric carcinoma MKN45 cells stably transfected with the full-length ST3GAL4 gene were characterised by glycomic and sialoproteomic analysis. Complementary strategies were applied to assess the glycomic alterations induced by ST3GAL4 overexpression. The N- and O-glycome data were generated in two......-MS/MS identification was performed. This analysis identified 47 proteins with significantly increased sialylation. The data in this article is associated with the research article published in Biochim Biophys Acta "Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine...... kinase activation in cancer" [1]....

Adhesion Regulating Molecule 1 Mediates HAP40 Overexpression-Induced Mitochondrial Defects

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Huang, Zih-Ning; Chung, Her Min; Fang, Su-Chiung; Her, Lu-Shiun

2017-01-01

Striatal neuron death in Huntington's disease is associated with abnormal mitochondrial dynamics and functions. However, the mechanisms for this mitochondrial dysregulation remain elusive. Increased accumulation of Huntingtin-associated protein 40 (HAP40) has been shown to be associated with Huntington's disease. However, the link between increased HAP40 and Huntington's disease remains largely unknown. Here we show that HAP40 overexpression causes mitochondrial dysfunction and reduces cell viability in the immortalized mouse striatal neurons. HAP40-associated mitochondrial dysfunction is associated with reduction of adhesion regulating molecule 1 (ADRM1) protein. Consistently, depletion of ADRM1 by shRNAs impaired mitochondrial functions and increased mitochondrial fragmentation in mouse striatal cells. Moreover, reducing ADRM1 levels enhanced activity of fission factor dynamin-related GTPase protein 1 (Drp1) via increased phosphorylation at serine 616 of Drp1 (Drp1Ser616). Restoring ADRM1 protein levels was able to reduce HAP40-induced ROS levels and mitochondrial fragmentation and improved mitochondrial functions and cell viability. Moreover, reducing Drp1 activity by Drp1 inhibitor, Mdivi-1, ameliorates both HAP40 overexpression- and ADRM1 depletion-induced mitochondrial dysfunction. Taken together, our studies suggest that HAP40-mediated reduction of ADRM1 alters the mitochondrial fission activity and results in mitochondrial fragmentation and mitochondrial dysfunction. PMID:29209146

HER2 overexpression elicits a proinflammatory IL-6 autocrine signaling loop that is critical for tumorigenesis.

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Hartman, Zachary C; Yang, Xiao-Yi; Glass, Oliver; Lei, Gangjun; Osada, Takuya; Dave, Sandeep S; Morse, Michael A; Clay, Timothy M; Lyerly, Herbert K

2011-07-01

HER2 overexpression occurs in approximately 25% of breast cancers, where it correlates with poor prognosis. Likewise, systemic inflammation in breast cancer correlates with poor prognosis, although the process is not understood. In this study, we explored the relationship between HER2 and inflammation, comparing the effects of overexpressing wild-type or mutated inactive forms of HER2 in primary human breast cells. Wild-type HER2 elicited a profound transcriptional inflammatory profile, including marked elevation of interleukin-6 (IL-6) expression, which we established to be a critical determinant of HER2 oncogenesis. Mechanistic investigations revealed that IL-6 secretion induced by HER2 overexpression activated Stat3 and altered gene expression, enforcing an autocrine loop of IL-6/Stat3 expression. Both mouse and human in vivo models of HER2-amplified breast carcinoma relied critically on this HER2-IL-6-Stat3 signaling pathway. Our studies offer the first direct evidence linking HER2 to a systemic inflammatory mechanism that orchestrates HER2-mediated tumor growth. We suggest that the HER2-IL-6-STAT3 signaling axis we have defined in breast cancer could prompt new therapeutic or prevention strategies for treatment of HER2-amplified cancers. ©2011 AACR.

Maternal immune activation leads to selective functional deficits in offspring parvalbumin interneurons.

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Canetta, S; Bolkan, S; Padilla-Coreano, N; Song, L J; Sahn, R; Harrison, N L; Gordon, J A; Brown, A; Kellendonk, C

2016-07-01

Abnormalities in prefrontal gamma aminobutyric acid (GABA)ergic transmission, particularly in fast-spiking interneurons that express parvalbumin (PV), are hypothesized to contribute to the pathophysiology of multiple psychiatric disorders, including schizophrenia, bipolar disorder, anxiety disorders and depression. While primarily histological abnormalities have been observed in patients and in animal models of psychiatric disease, evidence for abnormalities in functional neurotransmission at the level of specific interneuron populations has been lacking in animal models and is difficult to establish in human patients. Using an animal model of a psychiatric disease risk factor, prenatal maternal immune activation (MIA), we found reduced functional GABAergic transmission in the medial prefrontal cortex (mPFC) of adult MIA offspring. Decreased transmission was selective for interneurons expressing PV, resulted from a decrease in release probability and was not observed in calretinin-expressing neurons. This deficit in PV function in MIA offspring was associated with increased anxiety-like behavior and impairments in attentional set shifting, but did not affect working memory. Furthermore, cell-type specific optogenetic inhibition of mPFC PV interneurons was sufficient to impair attentional set shifting and enhance anxiety levels. Finally, we found that in vivo mPFC gamma oscillations, which are supported by PV interneuron function, were linearly correlated with the degree of anxiety displayed in adult mice, and that this correlation was disrupted in MIA offspring. These results demonstrate a selective functional vulnerability of PV interneurons to MIA, leading to affective and cognitive symptoms that have high relevance for schizophrenia and other psychiatric disorders.

Overexpression of SOS genes in ciprofloxacin resistant Escherichia coli mutants.

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Pourahmad Jaktaji, Razieh; Pasand, Shirin

2016-01-15

Fluoroquinolones are important antibiotics for the treatment of urinary tract infections caused by Escherichia coli. Mutational studies have shown that ciprofloxacin, a member of fluoroquinolones induces SOS response and mutagenesis in pathogenic bacteria which in turn develop antibiotic resistance. However, inhibition of SOS response can increase recombination activity which in turn leads to genetic variation. The aim of this study was to measure 5 SOS genes expressions in nine E. coli mutants with different MICs for ciprofloxacin following exposure to ciprofloxacin. Gene expression was assessed by quantitative real time PCR. Gene alteration assessment was conducted by PCR amplification and DNA sequencing. Results showed that the expression of recA was increased in 5 mutants. This overexpression is not related to gene alteration, and enhances the expression of polB and umuCD genes encoding nonmutagenic and mutagenic polymerases, respectively. The direct relationship between the level of SOS expression and the level of resistance to ciprofloxacin was also indicated. It was concluded that novel therapeutic strategy that inhibits RecA activity would enhance the efficiency of common antibiotics against pathogenic bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

Simultaneous Aurora-A/STK15 overexpression and centrosome amplification induce chromosomal instability in tumour cells with a MIN phenotype

International Nuclear Information System (INIS)

Lentini, Laura; Amato, Angela; Schillaci, Tiziana; Di Leonardo, Aldo

2007-01-01

Genetic instability is a hallmark of tumours and preneoplastic lesions. The predominant form of genome instability in human cancer is chromosome instability (CIN). CIN is characterized by chromosomal aberrations, gains or losses of whole chromosomes (aneuploidy), and it is often associated with centrosome amplification. Centrosomes control cell division by forming a bipolar mitotic spindle and play an essential role in the maintenance of chromosomal stability. However, whether centrosome amplification could directly cause aneuploidy is not fully established. Also, alterations in genes required for mitotic progression could be involved in CIN. A major candidate is represented by Aurora-A/STK15 that associates with centrosomes and is overexpressed in several types of human tumour. Centrosome amplification were induced by hydroxyurea treatment and visualized by immunofluorescence microscopy. Aurora-A/STK15 ectopic expression was achieved by retroviral infection and puromycin selection in HCT116 tumour cells. Effects of Aurora-A/STK15 depletion on centrosome status and ploidy were determined by Aurora-A/STK15 transcriptional silencing by RNA interference. Changes in the expression levels of some mitotic genes were determined by Real time RT-PCR. We investigated whether amplification of centrosomes and overexpression of Aurora-A/STK15 induce CIN using as a model system a colon carcinoma cell line (HCT116). We found that in HCT116 cells, chromosomally stable and near diploid cells harbouring a MIN phenotype, centrosome amplification induced by hydroxyurea treatment is neither maintained nor induces aneuploidy. On the contrary, ectopic overexpression of Aurora-A/STK15 induced supernumerary centrosomes and aneuploidy. Aurora-A/STK15 transcriptional silencing by RNA interference in cells ectopically overexpressing this kinase promptly decreased cell numbers with supernumerary centrosomes and aneuploidy. Our results show that centrosome amplification alone is not sufficient

Simultaneous Aurora-A/STK15 overexpression and centrosome amplification induce chromosomal instability in tumour cells with a MIN phenotype

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Schillaci Tiziana

2007-11-01

Full Text Available Abstract Background Genetic instability is a hallmark of tumours and preneoplastic lesions. The predominant form of genome instability in human cancer is chromosome instability (CIN. CIN is characterized by chromosomal aberrations, gains or losses of whole chromosomes (aneuploidy, and it is often associated with centrosome amplification. Centrosomes control cell division by forming a bipolar mitotic spindle and play an essential role in the maintenance of chromosomal stability. However, whether centrosome amplification could directly cause aneuploidy is not fully established. Also, alterations in genes required for mitotic progression could be involved in CIN. A major candidate is represented by Aurora-A/STK15 that associates with centrosomes and is overexpressed in several types of human tumour. Methods Centrosome amplification were induced by hydroxyurea treatment and visualized by immunofluorescence microscopy. Aurora-A/STK15 ectopic expression was achieved by retroviral infection and puromycin selection in HCT116 tumour cells. Effects of Aurora-A/STK15 depletion on centrosome status and ploidy were determined by Aurora-A/STK15 transcriptional silencing by RNA interference. Changes in the expression levels of some mitotic genes were determined by Real time RT-PCR. Results We investigated whether amplification of centrosomes and overexpression of Aurora-A/STK15 induce CIN using as a model system a colon carcinoma cell line (HCT116. We found that in HCT116 cells, chromosomally stable and near diploid cells harbouring a MIN phenotype, centrosome amplification induced by hydroxyurea treatment is neither maintained nor induces aneuploidy. On the contrary, ectopic overexpression of Aurora-A/STK15 induced supernumerary centrosomes and aneuploidy. Aurora-A/STK15 transcriptional silencing by RNA interference in cells ectopically overexpressing this kinase promptly decreased cell numbers with supernumerary centrosomes and aneuploidy. Conclusion Our

RCC2 over-expression in tumor cells alters apoptosis and drug sensitivity by regulating Rac1 activation.

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Wu, Nan; Ren, Dong; Li, Su; Ma, Wenli; Hu, Shaoyan; Jin, Yan; Xiao, Sheng

2018-01-10

Small GTP binding protein Rac1 is a component of NADPH oxidases and is essential for superoxide-induced cell death. Rac1 is activated by guanine nucleotide exchange factors (GEFs), and this activation can be blocked by regulator of chromosome condensation 2 (RCC2), which binds the switch regions of Rac1 to prevent access from GEFs. Three cancer cell lines with up- or down-regulation of RCC2 were used to evaluate cell proliferation, apoptosis, Rac1 signaling and sensitivity to a group of nine chemotherapeutic drugs. RCC2 expression in lung cancer and ovarian cancer were studied using immunochemistry stain of tumor tissue arrays. Forced RCC2 expression in tumor cells blocked spontaneous- or Staurosporine (STS)-induced apoptosis. In contrast, RCC2 knock down in these cells resulted in increased apoptosis to STS treatment. The protective activity of RCC2 on apoptosis was revoked by a constitutively activated Rac1, confirming a role of RCC2 in apoptosis by regulating Rac1. In an immunohistochemistry evaluation of tissue microarray, RCC2 was over-expressed in 88.3% of primary lung cancer and 65.2% of ovarian cancer as compared to non-neoplastic lung and ovarian tissues, respectively. Because chemotherapeutic drugs can kill tumor cells by activating Rac1/JNK pathway, we suspect that tumors with RCC2 overexpression would be more resistant to these drugs. Tumor cells with forced RCC2 expression indeed had significant difference in drug sensitivity compared to parental cells using a panel of common chemotherapeutic drugs. RCC2 regulates apoptosis by blocking Rac1 signaling. RCC2 expression in tumor can be a useful marker for predicting chemotherapeutic response.

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis.

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Cai, Dongbo; Wang, Hao; He, Penghui; Zhu, Chengjun; Wang, Qin; Wei, Xuetuan; Nomura, Christopher T; Chen, Shouwen

2017-04-24

Signal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases. In this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis. We observed dramatic decreases in the concentrations of both target proteins (45 and 49%, respectively) in a sppA deficient strain, while the extracellular protein yields of nattokinase and α-amylase were increased by 30 and 67% respectively in a strain overexpressing SppA. In addition, biomass, specific enzyme activities and the relative gene transcriptional levels were also enhanced due to the overexpression of sppA, while altering the expression levels of tepA had no effect on the concentrations of the secreted target proteins. Our results confirm that SppA, but not TepA, plays an important functional role for protein secretion in B. licheniformis. Our results indicate that the sppA overexpression strain, B. licheniformis BL10GS, could be used as a promising host strain for the industrial production of heterologous secreted proteins.

Characterization of Arabidopsis thaliana FLAVONOL SYNTHASE 1 (FLS1) -overexpression plants in response to abiotic stress.

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Nguyen, Nguyen Hoai; Kim, Jun Hyeok; Kwon, Jaeyoung; Jeong, Chan Young; Lee, Wonje; Lee, Dongho; Hong, Suk-Whan; Lee, Hojoung

2016-06-01

Flavonoids are an important group of secondary metabolites that are involved in plant growth and contribute to human health. Many studies have focused on the biosynthesis pathway, biochemical characters, and biological functions of flavonoids. In this report, we showed that overexpression of FLS1 (FLS1-OX) not only altered seed coat color (resulting in a light brown color), but also affected flavonoid accumulation. Whereas fls1-3 mutants accumulated higher anthocyanin levels, FLS1-OX seedlings had lower levels than those of the wild-type. Besides, shoot tissues of FLS1-OX plants exhibited lower flavonol levels than those of the wild-type. However, growth performance and abiotic stress tolerance of FLS1-OX, fls1-3, and wild-type plants were not significantly different. Taken together, FLS1 can be manipulated (i.e., silenced or overexpressed) to redirect the flavonoid biosynthetic pathway toward anthocyanin production without negative effects on plant growth and development. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Overexpression of Rac1 in leukemia patients and its role in leukemia cell migration and growth

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Wang, Jiying; Rao, Qing; Wang, Min; Wei, Hui; Xing, Haiyan; Liu, Hang; Wang, Yanzhong; Tang, Kejing; Peng, Leiwen; Tian, Zheng; Wang, Jianxiang

2009-01-01

Rac1 belongs to the Rho family that act as critical mediators of signaling pathways controlling cell migration and proliferation and contributes to the interactions of hematopoietic stem cells with their microenvironment. Alteration of Rac1 might result in unbalanced interactions and ultimately lead to leukemogenesis. In this study, we analyze the expression of Rac1 protein in leukemia patients and determine its role in the abnormal behaviours of leukemic cells. Rac1 protein is overexpressed in primary acute myeloid leukemia cells as compared to normal bone marrow mononuclear cells. siRNA-mediated silencing of Rac1 in leukemia cell lines induced inhibition of cell migration, proliferation, and colony formation. Additionally, blocking Rac1 activity by an inhibitor of Rac1-GTPase, NSC23766, suppressed cell migration and growth. We conclude that overexpression of Rac1 contributes to the accelerated migration and high proliferation potential of leukemia cells, which could be implicated in leukemia development and progression.

Overexpression of Rac1 in leukemia patients and its role in leukemia cell migration and growth

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Wang, Jiying [State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 288 Nanjing Road, Tianjin 300020 (China); Rao, Qing, E-mail: raoqing@gmail.com [State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 288 Nanjing Road, Tianjin 300020 (China); Wang, Min; Wei, Hui; Xing, Haiyan; Liu, Hang; Wang, Yanzhong; Tang, Kejing; Peng, Leiwen; Tian, Zheng; Wang, Jianxiang [State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 288 Nanjing Road, Tianjin 300020 (China)

2009-09-04

Rac1 belongs to the Rho family that act as critical mediators of signaling pathways controlling cell migration and proliferation and contributes to the interactions of hematopoietic stem cells with their microenvironment. Alteration of Rac1 might result in unbalanced interactions and ultimately lead to leukemogenesis. In this study, we analyze the expression of Rac1 protein in leukemia patients and determine its role in the abnormal behaviours of leukemic cells. Rac1 protein is overexpressed in primary acute myeloid leukemia cells as compared to normal bone marrow mononuclear cells. siRNA-mediated silencing of Rac1 in leukemia cell lines induced inhibition of cell migration, proliferation, and colony formation. Additionally, blocking Rac1 activity by an inhibitor of Rac1-GTPase, NSC23766, suppressed cell migration and growth. We conclude that overexpression of Rac1 contributes to the accelerated migration and high proliferation potential of leukemia cells, which could be implicated in leukemia development and progression.

Overexpression of mtDNA-associated AtWhy2 compromises mitochondrial function

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Abou-Rached Charbel

2008-04-01

Full Text Available Abstract Background StWhy1, a member of the plant-specific Whirly single-stranded DNA-binding protein family, was first characterized as a transcription factor involved in the activation of the nuclear PR-10a gene following defense-related stress in potato. In Arabidopsis thaliana, Whirlies have recently been shown to be primarily localized in organelles. Two representatives of the family, AtWhy1 and AtWhy3 are imported into plastids while AtWhy2 localizes to mitochondria. Their function in organelles is currently unknown. Results To understand the role of mitochondrial Whirlies in higher plants, we produced A. thaliana lines with altered expression of the atwhy2 gene. Organellar DNA immunoprecipitation experiments demonstrated that AtWhy2 binds to mitochondrial DNA. Overexpression of atwhy2 in plants perturbs mitochondrial function by causing a diminution in transcript levels and mtDNA content which translates into a low activity level of respiratory chain complexes containing mtDNA-encoded subunits. This lowered activity of mitochondria yielded plants that were reduced in size and had distorted leaves that exhibited accelerated senescence. Overexpression of atwhy2 also led to early accumulation of senescence marker transcripts in mature leaves. Inactivation of the atwhy2 gene did not affect plant development and had no detectable effect on mitochondrial morphology, activity of respiratory chain complexes, transcription or the amount of mtDNA present. This lack of phenotype upon abrogation of atwhy2 expression suggests the presence of functional homologues of the Whirlies or the activation of compensating mechanisms in mitochondria. Conclusion AtWhy2 is associated with mtDNA and its overexpression results in the production of dysfunctional mitochondria. This report constitutes the first evidence of a function for the Whirlies in organelles. We propose that they could play a role in the regulation of the gene expression machinery of organelles.

In vivo overexpression of tumstatin domains by tumor cells inhibits their invasive properties in a mouse melanoma model

International Nuclear Information System (INIS)

Pasco, Sylvie; Ramont, Laurent; Venteo, Lydie; Pluot, Michel; Maquart, Francois-Xavier; Monboisse, Jean-Claude

2004-01-01

Our previous studies demonstrated that a synthetic peptide encompassing residues 185-203 of the noncollagenous (NC1) domain of the α3 chain of type IV collagen, named tumstatin, inhibits in vitro melanoma cell proliferation and migration. In the present study, B16F1 melanoma cells were stably transfected to overexpress the complete tumstatin domain (Tum 1-232) or its C-terminal part, encompassing residues 185-203 (Tum 183-232). Tumstatin domain overexpression inhibited B16F1 in vitro cell proliferation, anchorage-independent growth, and invasive properties. For studying the in vivo effect of overexpression, representative clones were subcutaneously injected into the left side of C57BL6 mice. In vivo tumor growth was decreased by -60% and -56%, respectively, with B16F1 cells overexpressing Tum 1-232 or Tum 183-232 compared to control cells. This inhibitory effect was associated with a decrease of in vivo cyclin D1 expression. We also demonstrated that the overexpression of Tum 1-232 or Tum 183-232 induced an in vivo down-regulation of proteolytic cascades involving matrix metalloproteinases (MMPs), especially the production or activation of MMP-2, MMP-9, MMP-13, as well as MMP-14. The plasminogen activation system was also altered in tumors with a decrease of urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) and a strong increase of plasminogen activator inhibitor-1 (PAI-1). Collectively, our results demonstrate that tumstatin or its C-terminal antitumor fragment, Tum 183-232, inhibits in vivo melanoma progression by triggering an intracellular transduction pathway, which involves a cyclic AMP (cAMP)-dependent mechanism

Differential Receptive Field Properties of Parvalbumin and Somatostatin Inhibitory Neurons in Mouse Auditory Cortex.

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Li, Ling-Yun; Xiong, Xiaorui R; Ibrahim, Leena A; Yuan, Wei; Tao, Huizhong W; Zhang, Li I

2015-07-01

Cortical inhibitory circuits play important roles in shaping sensory processing. In auditory cortex, however, functional properties of genetically identified inhibitory neurons are poorly characterized. By two-photon imaging-guided recordings, we specifically targeted 2 major types of cortical inhibitory neuron, parvalbumin (PV) and somatostatin (SOM) expressing neurons, in superficial layers of mouse auditory cortex. We found that PV cells exhibited broader tonal receptive fields with lower intensity thresholds and stronger tone-evoked spike responses compared with SOM neurons. The latter exhibited similar frequency selectivity as excitatory neurons. The broader/weaker frequency tuning of PV neurons was attributed to a broader range of synaptic inputs and stronger subthreshold responses elicited, which resulted in a higher efficiency in the conversion of input to output. In addition, onsets of both the input and spike responses of SOM neurons were significantly delayed compared with PV and excitatory cells. Our results suggest that PV and SOM neurons engage in auditory cortical circuits in different manners: while PV neurons may provide broadly tuned feedforward inhibition for a rapid control of ascending inputs to excitatory neurons, the delayed and more selective inhibition from SOM neurons may provide a specific modulation of feedback inputs on their distal dendrites. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Overexpression of miR529a confers enhanced resistance to oxidative stress in rice (Oryza sativa L.).

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Yue, Erkui; Liu, Zhen; Li, Chao; Li, Yu; Liu, Qiuxiang; Xu, Jian-Hong

2017-07-01

Overexpressing miR529a can enhance oxidative stress resistance by targeting OsSPL2 and OsSPL14 genes that can regulate the expression of their downstream SOD and POD related genes. MicroRNAs are involved in the regulation of plant developmental and physiological processes, and their expression can be altered when plants suffered environment stresses, including salt, oxidative, drought and Cadmium. The expression of microRNA529 (miR529) can be induced under oxidative stress. However, its biological function under abiotic stress responses is still unclear. In this study, miR529a was overexpressed to investigate the function of miR529a under oxidative stress in rice. Our results demonstrated that the expression of miR529a can be induced by exogenous H 2 O 2 , and overexpressing miR529a can increase plant tolerance to high level of H 2 O 2 , resulting in increased seed germination rate, root tip cell viability, reduced leaf rolling rate and chlorophyll retention. The expression of oxidative stress responsive genes and the activities of superoxide dismutase (SOD) and peroxidase (POD) were increased in miR529a overexpression plant, which could help to reduce redundant reactive oxygen species (ROS). Furthermore, only OsSPL2 and OsSPL14 were targeted by miR529a in rice seedlings, repressing their expression in miR529aOE plants could lead to strengthen plant tolerance to oxidation stress. Our study provided the evidence that overexpression of miR529a could strengthen oxidation resistance, and its target genes OsSPL2 and OsSPL14 were responsible for oxidative tolerance, implied the manipulation of miR529a and its target genes regulation on H 2 O 2 related response genes could improve oxidative stress tolerance in rice.

Genetic and genomic analysis modeling of germline c-MYC overexpression and cancer susceptibility

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Nunes Virginia

2008-01-01

Full Text Available Abstract Background Germline genetic variation is associated with the differential expression of many human genes. The phenotypic effects of this type of variation may be important when considering susceptibility to common genetic diseases. Three regions at 8q24 have recently been identified to independently confer risk of prostate cancer. Variation at 8q24 has also recently been associated with risk of breast and colorectal cancer. However, none of the risk variants map at or relatively close to known genes, with c-MYC mapping a few hundred kilobases distally. Results This study identifies cis-regulators of germline c-MYC expression in immortalized lymphocytes of HapMap individuals. Quantitative analysis of c-MYC expression in normal prostate tissues suggests an association between overexpression and variants in Region 1 of prostate cancer risk. Somatic c-MYC overexpression correlates with prostate cancer progression and more aggressive tumor forms, which was also a pathological variable associated with Region 1. Expression profiling analysis and modeling of transcriptional regulatory networks predicts a functional association between MYC and the prostate tumor suppressor KLF6. Analysis of MYC/Myc-driven cell transformation and tumorigenesis substantiates a model in which MYC overexpression promotes transformation by down-regulating KLF6. In this model, a feedback loop through E-cadherin down-regulation causes further transactivation of c-MYC. Conclusion This study proposes that variation at putative 8q24 cis-regulator(s of transcription can significantly alter germline c-MYC expression levels and, thus, contribute to prostate cancer susceptibility by down-regulating the prostate tumor suppressor KLF6 gene.

Exposure to parvalbumin allergen and aerosols among herring processing workers.

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Dahlman-Höglund, Anna; Renström, Anne; Acevedo, Fernando; Andersson, Eva

2013-10-01

There are increasing reports of allergies and respiratory symptoms among workers in the fish processing industry, coinciding with an increasing use of high-pressure water in the processing plants. However, few studies have measured exposure in these work environments. The aim of this study was to characterize the occupational exposure of workers to herring antigen and to screen environmental factors at a herring (Clupea harengus) plant in which new and more encapsulated filleting machines had been installed. To assist in this, a method to assess airborne exposure to herring allergen was needed. Exposure to airborne herring antigen, mould spores, and endotoxin were measured during work. Antigen exposure was assessed using a newly developed sensitive (detection limit, 0.1 ng ml(-1)) rabbit polyclonal sandwich enzyme-linked immunosorbent assay against the major herring muscle protein allergen, parvalbumin. Aerosols were measured by mass concentration (DataRAM) and number of particles (Climet I-500). Personal geometric mean herring allergen exposure was 986 ng m(-3) at the old filleting workstations and 725 ng m(-3) at the new workstations (difference not significant). Outside the production room, the level was ~130 ng m(-3). Number of particles and mass concentration were both significantly lower around the new machines than around the old machines (P < 0.001 and P < 0.0001, respectively). The highest particle count was seen for the 0.3-0.5 μm fraction, with more than 400,000 particles per cubic metre air. Endotoxin concentration in the air varied between 3 and 92 EU m(-3), with the highest levels when the catch mainly contained herring that had eaten krill or seaweed. We developed a sensitive method to detect herring antigen. High exposure to herring antigen was measured during filleting work. The particles in the air around the fillet machines were mainly <0.5 μm and the newer encapsulated machines generated fewer particles. It is important to reduce occupational

HER2 overexpression elicits a pro-inflammatory IL-6 autocrine signaling loop that is critical for tumorigenesis

Science.gov (United States)

Hartman, Zachary C.; Yang, Xiao-Yi; Glass, Oliver; Lei, Gangjun; Osada, Takuya; Dave, Sandeep S.; Morse, Michael A.; Clay, Timothy M.; Lyerly, Herbert Kim

2011-01-01

HER2 overexpression occurs in ~25% of breast cancers where it correlates with poor prognosis. Likewise, systemic inflammation in breast cancer correlates with poor prognosis although the process is not understood. In this study, we explored the relationship between HER2 and inflammation, comparing the effects of overexpressing wild-type or mutated inactive forms of HER2 in primary human breast cells. Wild-type HER2 elicited a profound transcriptional inflammatory profile, including marked elevation of IL-6 expression, which we established to be a critical determinant of HER2 oncogenesis. Mechanistic investigations revealed that IL-6 secretion induced by HER2 overexpression activated Stat3 and altered gene expression, enforcing an autocrine loop of IL-6/Stat3 expression. Both mouse and human in vivo models of HER2 amplified breast carcinoma relied critically on this HER2-IL-6-Stat3 signaling pathway. Our studies offer the first direct evidence linking HER2 to a systemic inflammatory mechanism that orchestrates HER2-mediated tumor growth. We suggest that the HER2-IL6-STAT3 signaling axis we have defined in breast cancer could prompt new therapeutic or prevention strategies for treatment of HER2-amplified cancers. PMID:21518778

Overexpression of Enterococcus faecalis elr operon protects from phagocytosis.

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Cortes-Perez, Naima G; Dumoulin, Romain; Gaubert, Stéphane; Lacoux, Caroline; Bugli, Francesca; Martin, Rebeca; Chat, Sophie; Piquand, Kevin; Meylheuc, Thierry; Langella, Philippe; Sanguinetti, Maurizio; Posteraro, Brunella; Rigottier-Gois, Lionel; Serror, Pascale

2015-05-25

Mechanisms underlying the transition from commensalism to virulence in Enterococcus faecalis are not fully understood. We previously identified the enterococcal leucine-rich protein A (ElrA) as a virulence factor of E. faecalis. The elrA gene is part of an operon that comprises four other ORFs encoding putative surface proteins of unknown function. In this work, we compared the susceptibility to phagocytosis of three E. faecalis strains, including a wild-type (WT), a ΔelrA strain, and a strain overexpressing the whole elr operon in order to understand the role of this operon in E. faecalis virulence. While both WT and ΔelrA strains were efficiently phagocytized by RAW 264.7 mouse macrophages, the elr operon-overexpressing strain showed a decreased capability to be internalized by the phagocytic cells. Consistently, the strain overexpressing elr operon was less adherent to macrophages than the WT strain, suggesting that overexpression of the elr operon could confer E. faecalis with additional anti-adhesion properties. In addition, increased virulence of the elr operon-overexpressing strain was shown in a mouse peritonitis model. Altogether, our results indicate that overexpression of the elr operon facilitates the E. faecalis escape from host immune defenses.

Nanotized PPARα Overexpression Targeted to Hypertrophied Myocardium Improves Cardiac Function by Attenuating the p53-GSK3β-Mediated Mitochondrial Death Pathway.

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Rana, Santanu; Datta, Ritwik; Chaudhuri, Ratul Datta; Chatterjee, Emeli; Chawla-Sarkar, Mamta; Sarkar, Sagartirtha

2018-05-09

Metabolic remodeling of cardiac muscles during pathological hypertrophy is characterized by downregulation of fatty acid oxidation (FAO) regulator, peroxisome proliferator-activated receptor alpha (PPARα). Thereby, we hypothesized that a cardiac-specific induction of PPARα might restore the FAO-related protein expression and resultant energy deficit. In the present study, consequences of PPARα augmentation were evaluated for amelioration of chronic oxidative stress, myocyte apoptosis, and cardiac function during pathological cardiac hypertrophy. Nanotized PPARα overexpression targeted to myocardium was done by a stearic acid-modified carboxymethyl-chitosan (CMC) conjugated to a 20-mer myocyte-targeted peptide (CMCP). Overexpression of PPARα ameliorated pathological hypertrophy and improved cardiac function. Augmented PPARα in hypertrophied myocytes revealed downregulated p53 acetylation (lys 382), leading to reduced apoptosis. Such cells showed increased binding of PPARα with p53 that in turn reduced interaction of p53 with glycogen synthase kinase-3β (GSK3β), which upregulated inactive phospho-GSK3β (serine [Ser]9) expression within mitochondrial protein fraction. Altogether, the altered molecular milieu in PPARα-overexpressed hypertrophy groups restored mitochondrial structure and function both in vitro and in vivo. Cardiomyocyte-targeted overexpression of a protein of interest (PPARα) by nanotized plasmid has been described for the first time in this study. Our data provide a novel insight towards regression of pathological hypertrophy by ameliorating mitochondrial oxidative stress in targeted PPARα-overexpressed myocardium. PPARα-overexpression during pathological hypertrophy showed substantial betterment of mitochondrial structure and function, along with downregulated apoptosis. Myocardium-targeted overexpression of PPARα during pathological cardiac hypertrophy led to an overall improvement of cardiac energy deficit and subsequent cardiac

Parvalbumin immunoreactivity in the auditory cortex of a mouse model of presbycusis.

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Martin del Campo, H N; Measor, K R; Razak, K A

2012-12-01

Age-related hearing loss (presbycusis) affects ∼35% of humans older than sixty-five years. Symptoms of presbycusis include impaired discrimination of sounds with fast temporal features, such as those present in speech. Such symptoms likely arise because of central auditory system plasticity, but the underlying components are incompletely characterized. The rapid spiking inhibitory interneurons that co-express the calcium binding protein Parvalbumin (PV) are involved in shaping neural responses to fast spectrotemporal modulations. Here, we examined cortical PV expression in the C57bl/6 (C57) mouse, a strain commonly studied as a presbycusis model. We examined if PV expression showed auditory cortical field- and layer-specific susceptibilities with age. The percentage of PV-expressing cells relative to Nissl-stained cells was counted in the anterior auditory field (AAF) and primary auditory cortex (A1) in three age groups: young (1-2 months), middle-aged (6-8 months) and old (14-20 months). There were significant declines in the percentage of cells expressing PV at a detectable level in layers I-IV of both A1 and AAF in the old mice compared to young mice. In layers V-VI, there was an increase in the percentage of PV-expressing cells in the AAF of the old group. There were no changes in percentage of PV-expressing cells in layers V-VI of A1. These data suggest cortical layer(s)- and field-specific susceptibility of PV+ cells with presbycusis. The results are consistent with the hypothesis that a decline in inhibitory neurotransmission, particularly in the superficial cortical layers, occurs with presbycusis. Copyright © 2012 Elsevier B.V. All rights reserved.

Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

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Marizela Delic

2014-10-01

Full Text Available Oxidative folding of secretory proteins in the endoplasmic reticulum (ER is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

Bcl-2 overexpression: effects on transmembrane calcium movement

International Nuclear Information System (INIS)

Rangaswami, Arun A.; Premack, Brett; Walleczek, Jan; Killoran, Pamela; Gardner, Phyllis; Knox, Susan J.

1996-01-01

calcium influx rate was measured as above. Apoptosis was measured by morphological, DNA fragmentation, and FACS analyses. Levels of Bcl-2 expression in the various cell types were quantified by Western Blot Analysis. Results: In both cell lines, Bcl-2 overexpression had no effect on the resting cytosolic calcium concentration. However, there was a 1.7 to 1.8 fold increase in the cytosolic calcium concentration in the Bcl-2 transfected cells following exposure to 0.5 μM thapsigargin (p<0.001), which was not observed in similarly treated parental or neomycin control transfected cells. Furthermore, this difference was only observed in cells incubated in media containing 2 mM extracellular calcium and was abrogated by chelation of external calcium with EGTA. Quantification of calcium influx rates by the manganese quench technique demonstrated enhanced calcium influx rates in Bcl-2 transfected cell lines both at rest and in response to depletion of cellular calcium stores by thapsigargin. Furthermore, neither hyper polarization of the cells by Valinomycin nor depolarization by isotonic potassium chloride abrogated the relative enhancement of calcium entry in the Bcl-2 over expressing cell lines as compared with the parental or neomycin transfected controls. Conclusion: These preliminary results suggest that Bcl-2 overexpression may contribute to chemo and radioresistance of tumor cells by altering capacitive calcium entry. Although Bcl-2 overexpression does not alter the resting calcium concentration, it does significantly enhance the resting calcium influx rate, suggesting that the dynamic equilibrium of calcium movement across the plasma membrane is significantly altered in cells which over express the Bcl-2 onco-protein. This perturbation is independent of the effect of Bcl-2 on plasma membrane hyper polarization. Given the importance of calcium in signaling a variety of cellular processes including cell division and differentiation, these findings may help to explain the

Correlation of cytoplasmic beta-catenin and cyclin D1 overexpression during thyroid carcinogenesis around Semipalatinsk nuclear test site.

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Meirmanov, Serik; Nakashima, Masahiro; Kondo, Hisayoshi; Matsufuji, Reiko; Takamura, Noboru; Ishigaki, Katsu; Ito, Masahiro; Prouglo, Yuri; Yamashita, Shunichi; Sekine, Ichiro

2003-06-01

The Semipalatinsk nuclear test site (SNTS), the Republic of Kazakhstan, has been contaminated by radioactive fallout. The alteration of oncogenic molecules in thyroid cancer around the SNTS was considered worthy of analysis because it presented the potential to elucidate the relationship between radiation exposure and thyroid cancer. This study aimed to analyze both beta-catenin and cyclin D1 expressions in thyroid carcinomas around the SNTS. We examined nine cases of chronic thyroiditis, eight cases of follicular adenomas, and 23 cases of papillary carcinomas. Immunohistochemically, all carcinomas displayed a strong cytosolic beta-catenin expression, while both chronic thyroiditis and follicular adenomas showed a significantly lower cytoplasmic beta-catenin (22.2% and 37.5%, respectively). No cyclin D1 immunoreactivity was evident in chronic thyroiditis. In contrast, 62.5% of follicular adenomas and 87.0% of papillary carcinoma showed cyclin D1 overexpression. Additionally, a strong correlation between cytoplasmic beta-catenin and cyclin D1 expression was suggested in thyroid tumors. This study revealed a higher prevalence of both aberrant beta-catenin expression and cyclin D1 overexpression in papillary thyroid cancers around the SNTS than sporadic cases. The analysis of the alteration of the Wnt signaling-related molecules in thyroid cancer around the SNTS may be important to gain an insight into radiation-induced thyroid tumorigenesis.

PTK2B/Pyk2 overexpression improves a mouse model of Alzheimer's disease

KAUST Repository

Giralt, Albert; de Pins, Benoî t; Cifuentes-Dí az, Carmen; Ló pez-Molina, Laura; Farah, Amel Thamila; Tible, Marion; Deramecourt, Vincent; Arold, Stefan T.; Giné s, Silvia; Hugon, Jacques; Girault, Jean-Antoine

2018-01-01

and associated Src alterations. Overcoming this deficit by Pyk2 overexpression improved the behavioral and molecular phenotype of 5XFAD mice. Thus, our results in a mouse model of AD suggest that Pyk2 impairment may play a role in the symptoms of the disease.

PTK2B/Pyk2 overexpression improves a mouse model of Alzheimer's disease

KAUST Repository

Giralt, Albert

2018-05-24

autophosphorylation and associated Src alterations. Overcoming this deficit by Pyk2 overexpression improved the behavioral and molecular phenotype of 5XFAD mice. Thus, our results in a mouse model of AD suggest that Pyk2 impairment may play a role in the symptoms of the disease.

Over-expression of the mycobacterial trehalose-phosphate phosphatase OtsB2 results in a defect in macrophage phagocytosis associated with increased mycobacterial-macrophage adhesion

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Hao Li

2016-11-01

Full Text Available Trehalose-6-phosphate phosphatase (OtsB2 is involved in the OtsAB trehalose synthesis pathway to produce free trehalose and is strictly essential for mycobacterial growth. We wished to determine the effects of OtsB2 expression on mycobacterial phenotypes such as growth, phagocytosis and survival in macrophages. Mycobacterium bovis-BCG (BCG over-expressing OtsB2 were able to better survive in stationary phase. Over-expression of OtsB2 led to a decrease in phagocytosis but not survival in THP-1 macrophage-like cells, and this was not due to a decrease in general macrophage phagocytic activity. Surprisingly, when we investigated macrophage-mycobacterial interactions by flow cytometry and atomic force microscopy, we discovered that BCG over-expressing OtsB2 have stronger binding to THP-1 cells than wild-type BCG. These results suggest that altering OtsB2 expression has implications for mycobacterial host-pathogen interactions. Macrophage-mycobacteria phagocytic interactions are complex and merit further study.

Overexpression of histone demethylase Fbxl10 leads to enhanced migration in mouse embryonic fibroblasts

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Rohde, Magdalena; Sievers, Elisabeth; Janzer, Andreas [Institute of Pathology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn (Germany); Willmann, Dominica [Urologische Klinik/Frauenklinik und Zentrale Klinische Forschung, Klinikum der Universität Freiburg, Breisacherstrasse 66, 79106 Freiburg (Germany); Egert, Angela; Schorle, Hubert [Department of Developmental Pathology, Institute of Pathology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn (Germany); Schüle, Roland [Urologische Klinik/Frauenklinik und Zentrale Klinische Forschung, Klinikum der Universität Freiburg, Breisacherstrasse 66, 79106 Freiburg (Germany); Kirfel, Jutta, E-mail: Jutta.Kirfel@ukb.uni-bonn.de [Institute of Pathology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn (Germany)

2016-11-01

Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing, immune responses and invasive tumors all require the orchestrated movement of cells to specific locations. Histone demethylase proteins alter transcription by regulating the chromatin state at specific gene loci. FBXL10 is a conserved and ubiquitously expressed member of the JmjC domain-containing histone demethylase family and is implicated in the demethylation of H3K4me3 and H3K36me2 and thereby removing active chromatin marks. However, the physiological role of FBXL10 in vivo remains largely unknown. Therefore, we established an inducible gain of function model to analyze the role of Fbxl10 and compared wild-type with Fbxl10 overexpressing mouse embryonic fibroblasts (MEFs). Our study shows that overexpression of Fbxl10 in MEFs doesn’t influence the proliferation capability but leads to an enhanced migration capacity in comparison to wild-type MEFs. Transcriptome and ChIP-seq experiments demonstrated that Fbxl10 binds to genes involved in migration like Areg, Mdk, Lmnb1, Thbs1, Mgp and Cxcl12. Taken together, our results strongly suggest that Fbxl10 plays a critical role in migration by binding to the promoter region of migration-associated genes and thereby might influences cell behaviour to a possibly more aggressive phenotype. - Highlights: • Migration capability of MEFs is enhanced after Fbxl10 upregulation. • Overexpression of Fbxl10 induced migration-associated genes. • Fbxl10 binds directly to migration-associated genes.

Increase in cellulose accumulation and improvement of saccharification by overexpression of arabinofuranosidase in rice.

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Minako Sumiyoshi

Full Text Available Cellulosic biomass is available for the production of biofuel, with saccharification of the cell wall being a key process. We investigated whether alteration of arabinoxylan, a major hemicellulose in monocots, causes an increase in saccharification efficiency. Arabinoxylans have β-1,4-D-xylopyranosyl backbones and 1,3- or 1,4-α-l-arabinofuranosyl residues linked to O-2 and/or O-3 of xylopyranosyl residues as side chains. Arabinose side chains interrupt the hydrogen bond between arabinoxylan and cellulose and carry an ester-linked feruloyl substituent. Arabinose side chains are the base point for diferuloyl cross-links and lignification. We analyzed rice plants overexpressing arabinofuranosidase (ARAF to study the role of arabinose residues in the cell wall and their effects on saccharification. Arabinose content in the cell wall of transgenic rice plants overexpressing individual ARAF full-length cDNA (OsARAF1-FOX and OsARAF3-FOX decreased 25% and 20% compared to the control and the amount of glucose increased by 28.2% and 34.2%, respectively. We studied modifications of cell wall polysaccharides at the cellular level by comparing histochemical cellulose staining patterns and immunolocalization patterns using antibodies raised against α-(1,5-linked l-Ara (LM6 and β-(1,4-linked d-Xyl (LM10 and LM11 residues. However, they showed no visible phenotype. Our results suggest that the balance between arabinoxylan and cellulose might maintain the cell wall network. Moreover, ARAF overexpression in rice effectively leads to an increase in cellulose accumulation and saccharification efficiency, which can be used to produce bioethanol.

Overexpression of histone demethylase Fbxl10 leads to enhanced migration in mouse embryonic fibroblasts

International Nuclear Information System (INIS)

Rohde, Magdalena; Sievers, Elisabeth; Janzer, Andreas; Willmann, Dominica; Egert, Angela; Schorle, Hubert; Schüle, Roland; Kirfel, Jutta

2016-01-01

Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing, immune responses and invasive tumors all require the orchestrated movement of cells to specific locations. Histone demethylase proteins alter transcription by regulating the chromatin state at specific gene loci. FBXL10 is a conserved and ubiquitously expressed member of the JmjC domain-containing histone demethylase family and is implicated in the demethylation of H3K4me3 and H3K36me2 and thereby removing active chromatin marks. However, the physiological role of FBXL10 in vivo remains largely unknown. Therefore, we established an inducible gain of function model to analyze the role of Fbxl10 and compared wild-type with Fbxl10 overexpressing mouse embryonic fibroblasts (MEFs). Our study shows that overexpression of Fbxl10 in MEFs doesn’t influence the proliferation capability but leads to an enhanced migration capacity in comparison to wild-type MEFs. Transcriptome and ChIP-seq experiments demonstrated that Fbxl10 binds to genes involved in migration like Areg, Mdk, Lmnb1, Thbs1, Mgp and Cxcl12. Taken together, our results strongly suggest that Fbxl10 plays a critical role in migration by binding to the promoter region of migration-associated genes and thereby might influences cell behaviour to a possibly more aggressive phenotype. - Highlights: • Migration capability of MEFs is enhanced after Fbxl10 upregulation. • Overexpression of Fbxl10 induced migration-associated genes. • Fbxl10 binds directly to migration-associated genes.

Overexpression of Cholesteryl Ester Transfer Protein Increases Macrophage-Derived Foam Cell Accumulation in Atherosclerotic Lesions of Transgenic Rabbits

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Shoucui Gao

2017-01-01

Full Text Available High levels of plasma high-density lipoprotein-cholesterol (HDL-C are inversely associated with the risk of atherosclerosis and other cardiovascular diseases; thus, pharmacological inhibition of cholesteryl ester transfer protein (CETP is considered to be a therapeutic method of raising HDL-C levels. However, many CETP inhibitors have failed to achieve a clinical benefit despite raising HDL-C. In the study, we generated transgenic (Tg rabbits that overexpressed the human CETP gene to examine the influence of CETP on the development of atherosclerosis. Both Tg rabbits and their non-Tg littermates were fed a high cholesterol diet for 16 weeks. Plasma lipids and body weight were measured every 4 weeks. Gross lesion areas of the aortic atherosclerosis along with lesional cellular components were quantitatively analyzed. Overexpression of human CETP did not significantly alter the gross atherosclerotic lesion area, but the number of macrophages in lesions was significantly increased. Overexpression of human CETP did not change the plasma levels of total cholesterol or low-density lipoprotein cholesterol but lowered plasma HDL-C and increased triglycerides. These data revealed that human CETP may play an important role in the development of atherosclerosis mainly by decreasing HDL-C levels and increasing the accumulation of macrophage-derived foam cells.

Identification and overexpression of a Knotted1-like transcription factor in switchgrass (Panicum virgatum L. for lignocellulosic feedstock improvement

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Wegi eWuddineh

2016-04-01

Full Text Available High biomass production and wide adaptation has made switchgrass (Panicum virgatum L. an important candidate lignocellulosic bioenergy crop. One major limitation of this and other lignocellulosic feedstocks is the recalcitrance of complex carbohydrates to hydrolysis for conversion to biofuels. Lignin is the major contributor to recalcitrance as it limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars. Therefore, genetic manipulation of the lignin biosynthesis pathway is one strategy to reduce recalcitrance. Here, we identified a switchgrass Knotted1 transcription factor, PvKN1, with the aim of genetically engineering switchgrass for reduced biomass recalcitrance for biofuel production. Gene expression of the endogenous PvKN1 gene was observed to be highest in young inflorescences and stems. Ectopic overexpression of PvKN1 in switchgrass altered growth, especially in early developmental stages. Transgenic lines had reduced expression of most lignin biosynthetic genes accompanied by a reduction in lignin content suggesting the involvement of PvKN1 in the broad regulation of the lignin biosynthesis pathway. Moreover, the reduced expression of the Gibberellin 20-oxidase (GA20ox gene in tandem with the increased expression of Gibberellin 2-oxidase (GA2ox genes in transgenic PvKN1 lines suggest that PvKN1 may exert regulatory effects via modulation of GA signalling. Furthermore, overexpression of PvKN1 altered the expression of cellulose and hemicellulose biosynthetic genes and increased sugar release efficiency in transgenic lines. Our results demonstrated that switchgrass PvKN1 is a putative ortholog of maize KN1 that is linked to plant lignification and cell wall and development traits as a major regulatory gene. Therefore, targeted overexpression of PvKN1 in bioenergy feedstocks may provide one feasible strategy for reducing biomass recalcitrance and simultaneously improving plant growth characteristics.

Altered AIB1 or AIB1Δ3 Expression Impacts ERα Effects on Mammary Gland Stromal and Epithelial Content

Science.gov (United States)

Nakles, Rebecca E.; Shiffert, Maddalena Tilli; Díaz-Cruz, Edgar S.; Cabrera, M. Carla; Alotaiby, Maram; Miermont, Anne M.; Riegel, Anna T.

2011-01-01

Amplified in breast cancer 1 (AIB1) (also known as steroid receptor coactivator-3) is a nuclear receptor coactivator enhancing estrogen receptor (ER)α and progesterone receptor (PR)-dependent transcription in breast cancer. The splice variant AIB1Δ3 demonstrates increased ability to promote ERα and PR-dependent transcription. Both are implicated in breast cancer risk and antihormone resistance. Conditional transgenic mice tested the in vivo impact of AIB1Δ3 overexpression compared with AIB1 on histological features of increased breast cancer risk and growth response to estrogen and progesterone in the mammary gland. Combining expression of either AIB1 or AIB1Δ3 with ERα overexpression, we investigated in vivo cooperativity. AIB1 and AIB1Δ3 overexpression equivalently increased the prevalence of hyperplastic alveolar nodules but not ductal hyperplasia or collagen content. When AIB1 or AIB1Δ3 overexpression was combined with ERα, both stromal collagen content and ductal hyperplasia prevalence were significantly increased and adenocarcinomas appeared. Overexpression of AIB1Δ3, especially combined with overexpressed ERα, led to an abnormal response to estrogen and progesterone with significant increases in stromal collagen content and development of a multilayered mammary epithelium. AIB1Δ3 overexpression was associated with a significant increase in PR expression and PR downstream signaling genes. AIB1 overexpression produced less marked growth abnormalities and no significant change in PR expression. In summary, AIB1Δ3 overexpression was more potent than AIB1 overexpression in increasing stromal collagen content, inducing abnormal mammary epithelial growth, altering PR expression levels, and mediating the response to estrogen and progesterone. Combining ERα overexpression with either AIB1 or AIB1Δ3 overexpression augmented abnormal growth responses in both epithelial and stromal compartments. PMID:21292825

[Overexpression of FKS1 to improve yeast autolysis-stress].

Science.gov (United States)

Li, Jia; Wang, Jinjing; Li, Qi

2015-09-01

With the development of high gravity brewing, yeast cells are exposed to multiple brewing-associated stresses, such as increased osmotic pressure, enhanced alcohol concentration and nutritional imbalance. These will speed up yeast autolysis, which seriously influence beer flavor and quality. To increase yeast anti-autolytic ability, FKS1 overexpression strain was constructed by 18S rDNA. The concentration of β-1,3-glucan of overexpression strain was 62% higher than that of wild type strain. Meantime, FKS1 overexpression strain increased anti-stress ability at 8% ethanol, 0.4 mol/L NaCl and starvation stress. Under simulated autolysis, FKS1 showed good anti-autolytic ability by slower autolysis. These results confirms the potential of FKS1 overexpression to tackle yeast autolysis in high-gravity brewing.

Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus

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D.M.O. Bonci

2006-03-01

Full Text Available To quantify the effects of methylmercury (MeHg on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus at two dose levels (2 and 6 µg/g, ip. The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR and alphaPKC (alphaPKC-IR in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 µg/g showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 ± 393 cells/mm² compared to control (1886 ± 892 cells/mm²; P < 0.001. The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 ± 56 cells/mm² (2 µg/g and 845 ± 82 cells/mm² (6 µg/g, also lower than control (1312 ± 31 cells/mm²; P < 0.05, differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 µg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) – a class-I KNOX gene in potato

Science.gov (United States)

Mahajan, Ameya S.; Kondhare, Kirtikumar R.; Rajabhoj, Mohit P.; Kumar, Amit; Ghate, Tejashree; Ravindran, Nevedha; Habib, Farhat; Siddappa, Sundaresha; Banerjee, Anjan K.

2016-01-01

Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato. PMID:27217546

MET overexpression, gene amplification and relevant clinicopathological features in gastric adenocarcinoma.

Science.gov (United States)

Zhang, Jing; Guo, Lei; Liu, Xiuyun; Li, Wenbin; Ying, Jianming

2017-02-07

This study was conducted to investigate the expression of MET in Chinese gastric adenocarcinoma cohort, the correlation between MET overexpression and clinical pathological features, HER2 expression and MET gene amplification. A total of 816 gastric adenocarcinoma patients were included and MET and HER2 immunohistochemical (IHC) staining were performed. IHC and dual-color silver in situ hybridization analysis were performed in the tissue microarrays, constructed from the 240 patients who were randomly selected. MET overexpression (IHC 3+) was observed in 6.0% (49/816) of the cohort. MET overexpression rate was higher in patients with poor prognostic factors, such as clinical stages III/IV (p =0.012) and pathologic stages T3/T4 (p =0.027). The HER2 overexpression (IHC 3+) rate was 8.8% (72/816) and MET overexpression rate was higher in HER2 positive patients (9.7%, 7/72). A high concordance rate (94.6%) between MET overexpression and gene amplification was demonstrated. Therefore, MET overexpression could serve as a prognostic biomarker and a potential therapeutic target for gastric cancer.

Over-expression of KdSOC1 gene affected plantlet morphogenesis in Kalanchoe daigremontiana.

Science.gov (United States)

Zhu, Chen; Wang, Li; Chen, Jinhua; Liu, Chenglan; Zeng, Huiming; Wang, Huafang

2017-07-17

Kalanchoe daigremontiana reproduces asexually by producing plantlets along the leaf margin. The aim of this study was to identify the function of the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 gene in Kalanchoe daigremontiana (KdSOC1) during plantlet morphogenesis. In this study, KdSOC1 gene expression was detected at stem cell niche during in vitro somatic embryogenesis and plantlet morphogenesis. Disrupting endogenous auxin transportation suppressed the KdSOC1 gene response. Knockdown of the KdSOC1 gene caused a defect in cotyledon formation during the early heart stage of somatic embryogenesis. Over-expression (OE) of the KdSOC1 gene resulted in asymmetric plantlet distribution, a reduced number of plantlets, thicker leaves, and thicker vascular fibers. Higher KdPIN1 gene expression and auxin content were found in OE plant compared to those of wild-type plant leaves, which indicated possible KdSOC1 gene role in affecting auxin distribution and accumulation. KdSOC1 gene OE in DR5-GUS Arabidopsis reporting lines resulted in an abnormal auxin response pattern during different stages of somatic embryogenesis. In summary, the KdSOC1 gene OE might alter auxin distribution and accumulation along leaf margin to initiate plantlet formation and distribution, which is crucial for plasticity during plantlet formation under various environmental conditions.

Hand1 overexpression inhibits medulloblastoma metastasis

Energy Technology Data Exchange (ETDEWEB)

Asuthkar, Swapna; Guda, Maheedhara R. [Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Martin, Sarah E. [Department of Pathology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Antony, Reuben; Fernandez, Karen [Department of Pediatrics, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Lin, Julian [Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Tsung, Andrew J. [Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Illinois Neurological Institute, Peoria, IL 61656 (United States); Velpula, Kiran K., E-mail: velpula@uic.edu [Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States); Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (United States)

2016-08-19

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current treatment includes surgery, radiation and chemotherapy. However, ongoing treatment in patients is further classified according to the presence or absence of metastasis. Since metastatic medulloblastoma are refractory to current treatments, there is need to identify novel biomarkers that could be used to reduce metastatic potential, and more importantly be targeted therapeutically. Previously, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. We further demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand1. Among the histological subtypes classical and desmoplastic subtypes account for the majority while large cell/anaplastic variant is most commonly associated with metastatic disease. In this present study using immunohistochemical approach and patient data mining for the first time, we demonstrated that Hand1 expression is observed to be downregulated in all the subtypes of medulloblastoma. Previously we showed that Hand1 overexpression regulated medulloblastoma angiogenesis and here we investigated the role of Hand1 in the context of Epithelial-Mesenchymal Transition (EMT). Moreover, UW228 and D283 cells overexpressing Hand1 demonstrated decreased-expression of mesenchymal markers (N-cadherin, β-catenin and SOX2); metastatic marker (SMA); and increased expression of epithelial marker (E-cadherin). Strikingly, human pluripotent stem cell antibody array showed that Hand1 overexpression resulted in substantial decrease in pluripotency markers (Nanog, Oct3/4, Otx2, Flk1) suggesting that Hand1 expression may be essential to attenuate the EMT and our findings underscore a novel role for Hand1 in medulloblastoma metastasis. - Highlights: • Hand1 expression is downregulated in Medulloblastoma. • Hand1 over expression reduce

Hand1 overexpression inhibits medulloblastoma metastasis

International Nuclear Information System (INIS)

Asuthkar, Swapna; Guda, Maheedhara R.; Martin, Sarah E.; Antony, Reuben; Fernandez, Karen; Lin, Julian; Tsung, Andrew J.; Velpula, Kiran K.

2016-01-01

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current treatment includes surgery, radiation and chemotherapy. However, ongoing treatment in patients is further classified according to the presence or absence of metastasis. Since metastatic medulloblastoma are refractory to current treatments, there is need to identify novel biomarkers that could be used to reduce metastatic potential, and more importantly be targeted therapeutically. Previously, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. We further demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand1. Among the histological subtypes classical and desmoplastic subtypes account for the majority while large cell/anaplastic variant is most commonly associated with metastatic disease. In this present study using immunohistochemical approach and patient data mining for the first time, we demonstrated that Hand1 expression is observed to be downregulated in all the subtypes of medulloblastoma. Previously we showed that Hand1 overexpression regulated medulloblastoma angiogenesis and here we investigated the role of Hand1 in the context of Epithelial-Mesenchymal Transition (EMT). Moreover, UW228 and D283 cells overexpressing Hand1 demonstrated decreased-expression of mesenchymal markers (N-cadherin, β-catenin and SOX2); metastatic marker (SMA); and increased expression of epithelial marker (E-cadherin). Strikingly, human pluripotent stem cell antibody array showed that Hand1 overexpression resulted in substantial decrease in pluripotency markers (Nanog, Oct3/4, Otx2, Flk1) suggesting that Hand1 expression may be essential to attenuate the EMT and our findings underscore a novel role for Hand1 in medulloblastoma metastasis. - Highlights: • Hand1 expression is downregulated in Medulloblastoma. • Hand1 over expression reduce

Breast cancer cell cyclooxygenase-2 expression alters extracellular matrix structure and function and numbers of cancer associated fibroblasts.

Science.gov (United States)

Krishnamachary, Balaji; Stasinopoulos, Ioannis; Kakkad, Samata; Penet, Marie-France; Jacob, Desmond; Wildes, Flonne; Mironchik, Yelena; Pathak, Arvind P; Solaiyappan, Meiyappan; Bhujwalla, Zaver M

2017-03-14

Cyclooxygenase-2 (COX-2) is a critically important mediator of inflammation that significantly influences tumor angiogenesis, invasion, and metastasis. We investigated the role of COX-2 expressed by triple negative breast cancer cells in altering the structure and function of the extracellular matrix (ECM). COX-2 downregulation effects on ECM structure and function were investigated using magnetic resonance imaging (MRI) and second harmonic generation (SHG) microscopy of tumors derived from triple negative MDA-MB-231 breast cancer cells, and a derived clone stably expressing a short hairpin (shRNA) molecule downregulating COX-2. MRI of albumin-GdDTPA was used to characterize macromolecular fluid transport in vivo and SHG microscopy was used to quantify collagen 1 (Col1) fiber morphology. COX-2 downregulation decreased Col1 fiber density and altered macromolecular fluid transport. Immunohistochemistry identified significantly fewer activated cancer associated fibroblasts (CAFs) in low COX-2 expressing tumors. Metastatic lung nodules established by COX-2 downregulated cells were infrequent, smaller, and contained fewer Col1 fibers.COX-2 overexpression studies were performed with tumors derived from triple negative SUM-149 breast cancer cells lentivirally transduced to overexpress COX-2. SHG microscopy identified significantly higher Col1 fiber density in COX-2 overexpressing tumors with an increase of CAFs. These data expand upon the roles of COX-2 in shaping the structure and function of the ECM in primary and metastatic tumors, and identify the potential role of COX-2 in modifying the number of CAFs in tumors that may have contributed to the altered ECM.

Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins.

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Chunxiang Yao

Full Text Available Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2, react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat, an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a 'Tandem Mass Tag' (TMT labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.

Overexpression of Rice Wall-Associated Kinase 25 (OsWAK25) Alters Resistance to Bacterial and Fungal Pathogens

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Harkenrider, Mitch; Sharma, Rita; De Vleesschauwer, David; Tsao, Li; Zhang, Xuting; Chern, Mawsheng; Canlas, Patrick; Zuo, Shimin; Ronald, Pamela C.

2016-01-01

Wall-associated kinases comprise a sub-family of receptor-like kinases that function in plant growth and stress responses. Previous studies have shown that the rice wall-associated kinase, OsWAK25, interacts with a diverse set of proteins associated with both biotic and abiotic stress responses. Here, we show that wounding and BTH treatments induce OsWAK25 transcript expression in rice. We generated OsWAK25 overexpression lines and show that these lines exhibit a lesion mimic phenotype and enhanced expression of rice NH1 (NPR1 homolog 1), OsPAL2, PBZ1 and PR10. Furthermore, these lines show resistance to the hemibiotrophic pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae, yet display increased susceptibility to necrotrophic fungal pathogens, Rhizoctonia solani and Cochliobolus miyabeanus. PMID:26795719

Overexpression of DJ-1/PARK7, the Parkinson's disease-related protein, improves mitochondrial function via Akt phosphorylation on threonine 308 in dopaminergic neuron-like cells.

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Zhang, Yi; Gong, Xiao-Gang; Wang, Zhen-Zhen; Sun, Hong-Mei; Guo, Zhen-Yu; Hu, Jing-Hong; Ma, Ling; Li, Ping; Chen, Nai-Hong

2016-05-01

DJ-1/PARK7, the Parkinson's disease-related protein, plays an important role in mitochondrial function. However, the mechanisms by which DJ-1 affects mitochondrial function are not fully understood. Akt is a promoter of neuron survival and is partly involved in the neurodegenerative process. This research aimed at investigating a possible relationship between DJ-1 and Akt signalling in regulating mitochondrial function in the dopaminergic neuron-like cells SH-SY5Y and PC-12. Overexpression of DJ-1 was firstly validated at both the transcriptional and translational levels after transit transfection with plasmid pcDNA3-Flag-DJ-1. Confocal fluorescence microscopy demonstrated that overexpression of DJ-1 increased the mitochondrial mass, but did not disrupt the mitochondrial morphology. In addition, mitochondrial complex I activity was raised in DJ-1-overexpressing cells, and this rise occurred with an increase in cellular adenosine 5'-triphosphate content. Moreover, immunoblotting demonstrated that the levels of phosphoinositide 3-kinase and the total Akt were not altered in DJ-1-overexpressing cells, and nor was the Akt phosphorylation on serine 473 changed. By contrast, Akt phosphorylation on threonine 308 was significantly augmented by overexpression of DJ-1, and the expression of glycogen synthase kinase-3beta, a downstream effector of Akt, was suppressed. In summary, these results suggest that overexpression of DJ-1 improves the mitochondrial function, at least in part, through a mechanism involving Akt phosphorylation on threonine 308. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Overexpression of adenosine A2A receptors in rats: effects on depression, locomotion and anxiety

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Joana E Coelho

2014-06-01

Full Text Available Adenosine A2A receptors (A2AR are a sub-type of receptors enriched in basal ganglia, activated by the neuromodulator adenosine, which interact with dopamine D2 receptors. Although this reciprocal antagonistic interaction is well established in motor function, the outcome in dopamine-related behaviors remains uncertain, in particular in depression and anxiety. We have demonstrated an upsurge of A2AR associated to aging and chronic stress. Furthermore, Alzheimer’s disease patients present A2AR accumulation in cortical areas together with depressive signs. We now tested the impact of overexpressing A2AR in forebrain neurons on dopamine related behavior, namely depression. Adult male rats overexpressing human A2AR under the control of CaMKII promoter [Tg(CaMKII-hA2AR] and aged-matched wild-types (WT of the same strain (Sprague-Dawley were studied. The forced swimming test (FST, sucrose preference test (SPT and the open-field test (OFT were performed to evaluate behavioral despair, anhedonia, locomotion and anxiety. Tg(CaMKII-hA2AR animals spent more time floating and less time swimming in the FST and presented a decreased sucrose preference at 48h in the SPT. They also covered higher distances in the OFT and spent more time in the central zone than the WT. The results indicate that Tg(CaMKII-hA2AR rats exhibit depressive-like behavior, hyperlocomotion and altered exploratory behavior. This A2AR overexpression may explain the depressive signs found in aging, chronic stress and Alzheimer’s disease.

Overexpression of Adenosine A2A Receptors in Rats: Effects on Depression, Locomotion, and Anxiety.

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Coelho, Joana E; Alves, Pedro; Canas, Paula M; Valadas, Jorge S; Shmidt, Tatiana; Batalha, Vânia L; Ferreira, Diana G; Ribeiro, Joaquim A; Bader, Michael; Cunha, Rodrigo A; do Couto, Frederico Simões; Lopes, Luísa V

2014-01-01

Adenosine A2A receptors (A2AR) are a sub-type of receptors enriched in basal ganglia, activated by the neuromodulator adenosine, which interact with dopamine D2 receptors. Although this reciprocal antagonistic interaction is well-established in motor function, the outcome in dopamine-related behaviors remains uncertain, in particular in depression and anxiety. We have demonstrated an upsurge of A2AR associated to aging and chronic stress. Furthermore, Alzheimer's disease patients present A2AR accumulation in cortical areas together with depressive signs. We now tested the impact of overexpressing A2AR in forebrain neurons on dopamine-related behavior, namely depression. Adult male rats overexpressing human A2AR under the control of CaMKII promoter [Tg(CaMKII-hA2AR)] and aged-matched wild-types (WT) of the same strain (Sprague-Dawley) were studied. The forced swimming test (FST), sucrose preference test (SPT), and the open-field test (OFT) were performed to evaluate behavioral despair, anhedonia, locomotion, and anxiety. Tg(CaMKII-hA2AR) animals spent more time floating and less time swimming in the FST and presented a decreased sucrose preference at 48 h in the SPT. They also covered higher distances in the OFT and spent more time in the central zone than the WT. The results indicate that Tg(CaMKII-hA2AR) rats exhibit depressive-like behavior, hyperlocomotion, and altered exploratory behavior. This A2AR overexpression may explain the depressive signs found in aging, chronic stress, and Alzheimer's disease.

Purification and characterization of parvalbumins, the major allergens in red stingray (Dasyatis akajei).

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Cai, Qiu-Feng; Liu, Guang-Ming; Li, Teng; Hara, Kenji; Wang, Xi-Chang; Su, Wen-Jin; Cao, Min-Jie

2010-12-22

Fish has received increasing attention because it induces IgE-mediated food allergy. Parvalbumin (PV) represents the major allergen of fish, and IgE cross-reactivity to PV in various teleost fish species has been shown, while little information is available about allergens in elasmobranch fish. In this study, two PV isoforms (named as PV-I and PV-II) from red stingray (Dasyatis akajei) were purified to homogeneity by a series of procedures including ammonium sulfate precipitation and column chromatographies of DEAE-Sepharose and Sephacryl S-200. Purified PVs revealed a single band on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular masses of PV-I and PV-II were 12.29 and 11.95 kDa, respectively, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot using antifrog PV monoclonal antibody (PARV-19) showed positive reactions to the two proteins, confirming that they were PVs, although their immunological reactivities were weaker than those of PV from silver carp. The N-terminal amino acid sequence of PV-I was determined, and comparison with PVs from other fish species showed low homology between teleost and elasmobranch fish. The isoelectric points of PV-I and PV-II were 5.4 and 5.0, respectively, as determined by two-dimensional electrophoresis (2-DE), suggesting that both isoforms belong to the α-group. IgE immunoblotting analysis showed that sera from fish-allergic patients reacted to both PV-I and PV-II from red stingray. Thermal stability revealed that PV-I easily formed oligomers than PV-II, which might contribute to the maintenance of its allerginicity during heat processing.

Selenium status and over-expression of interleukin-15 in celiac disease and autoimmune thyroid diseases

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Anna Velia Stazi

2010-12-01

Full Text Available In celiac disease (CD, for its multifactorial nature, the target organs are not limited to the gut, but include thyroid, liver, skin and reproductive and nervous systems. Between the extraintestinal symptoms associated with CD, autoimmune thyroid diseases (AITDs are more evident, underlining as CD-related autoimmune alterations can be modulated not only by gluten but also by various concurrent endogenous (genetic affinity, over-expression of cytokines and exogenous (environment, nutritional deficiency factors. In their pathogenesis a central role for over-expression of interleukin-15 (IL-15 is shown, by inhibiting apoptosis, leading to the perpetuation of inflammation and tissue destruction. Thyroid is particularly sensitive to selenium deficiency because selenoproteins are significant in biosynthesis and activity of thyroid hormones; besides, some selenoproteins as glutathione peroxidase are involved in inhibiting apoptosis. Thus, selenium malabsorption in CD can be thought as a key factor directly leading to thyroid and intestinal damage. Considering the complexity of this interaction and on the basis of available evidence, the aim of this review is to assess as preventive and therapeutic target the role of IL-15 and selenium in the pathogeneses of both CD and AITD.

Prognostic implication of aquaporin 1 overexpression in resected lung adenocarcinoma.

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Bellezza, Guido; Vannucci, Jacopo; Bianconi, Fortunato; Metro, Giulio; Del Sordo, Rachele; Andolfi, Marco; Ferri, Ivana; Siccu, Paola; Ludovini, Vienna; Puma, Francesco; Sidoni, Angelo; Cagini, Lucio

2017-12-01

Aquaporins (AQPs) are a group of transmembrane water-selective channel proteins thought to play a role in the regulation of water permeability for plasma membranes. Indeed, high AQP levels have been suggested to promote the progression, invasion and metastasis of tumours. Specifically, AQP1 and AQP5 overexpression in lung adenocarcinoma (AC) have been suggested to be involved in molecular mechanisms in lung cancer. The aim of this retrospective cohort single-centre study was to assess both the levels of expression and therein the prognostic significance, regarding outcome of AQP1 and AQP5 in resected AC patients. Patients with histological diagnoses of lung AC submitted to pulmonary resection were included in this cohort study. Tissue microarrays containing cores from 185 ACs were prepared. AQP1 and AQP5 expressions were assessed by immunohistochemistry. Results were scored as either low (Score 0-2) or high (Score 3-9). Clinical data, pathological tumour-node-metastasis staging and follow-up were recorded. Multivariate Cox survival analysis and Fisher's t-test were performed. AQP1 overexpression was detected in 85 (46%) patients, while AQP5 overexpression was observed in 45 (24%) patients. AQP1 did not result being significantly correlated with clinical and pathological parameters, while AQP5 resulted more expressed in AC with mucinous and papillary predominant patterns. Patients with AQP1 overexpression had shorter disease-free survival (P = 0.001) compared with patients without AQP1 overexpression. Multivariate analysis confirmed that AQP1 overexpression was significantly associated with shorter disease-free survival (P = 0.001). Our results evidenced that AQP1 overexpression resulted in a shorter disease-free survival in lung AC patients. Being so, AQP1 overexpression might be an important prognostic marker in lung AC. © The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights

Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species

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Yu, Suyeun [Department of Preventive Medicine, College of Medicine, Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 136-705 (Korea, Republic of); Jang, Yeogil; Paik, Donggi [Department of Physiology, College of Medicine, Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 136-705 (Korea, Republic of); Lee, Eunil, E-mail: eunil@korea.ac.kr [Department of Preventive Medicine, College of Medicine, Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 136-705 (Korea, Republic of); Park, Joong-Jean, E-mail: parkjj@korea.ac.kr [Department of Physiology, College of Medicine, Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 136-705 (Korea, Republic of)

2015-10-02

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is a bifunctional enzyme involved in folate-dependent metabolism and highly expressed in rapidly proliferating cells. However, Nmdmc physiological roles remain unveiled. We found that ubiquitous Nmdmc overexpression enhanced Drosophila lifespan and stress resistance. Interestingly, Nmdmc overexpression in the fat body was sufficient to increase lifespan and tolerance against oxidative stress. In addition, these conditions coincided with significant decreases in the levels of mitochondrial ROS and Hsp22 as well as with a significant increase in the copy number of mitochondrial DNA. These results suggest that Nmdmc overexpression should be beneficial for mitochondrial homeostasis and increasing lifespan. - Highlights: • Ubiquitous Nmdmc overexpression enhanced lifespan and stress tolerance. • Nmdmc overexpression in the fat body extended longevity. • Fat body-specific Nmdmc overexpression increased oxidative stress resistance. • Nmdmc overexpression decreased Hsp22 transcript levels and ROS. • Nmdmc overexpression increased mitochondrial DNA copy number.

Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species

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Yu, Suyeun; Jang, Yeogil; Paik, Donggi; Lee, Eunil; Park, Joong-Jean

2015-01-01

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is a bifunctional enzyme involved in folate-dependent metabolism and highly expressed in rapidly proliferating cells. However, Nmdmc physiological roles remain unveiled. We found that ubiquitous Nmdmc overexpression enhanced Drosophila lifespan and stress resistance. Interestingly, Nmdmc overexpression in the fat body was sufficient to increase lifespan and tolerance against oxidative stress. In addition, these conditions coincided with significant decreases in the levels of mitochondrial ROS and Hsp22 as well as with a significant increase in the copy number of mitochondrial DNA. These results suggest that Nmdmc overexpression should be beneficial for mitochondrial homeostasis and increasing lifespan. - Highlights: • Ubiquitous Nmdmc overexpression enhanced lifespan and stress tolerance. • Nmdmc overexpression in the fat body extended longevity. • Fat body-specific Nmdmc overexpression increased oxidative stress resistance. • Nmdmc overexpression decreased Hsp22 transcript levels and ROS. • Nmdmc overexpression increased mitochondrial DNA copy number.

Overexpression of a flower-specific aerolysin-like protein from the dioecious plant Rumex acetosa alters flower development and induces male sterility in transgenic tobacco.

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Manzano, Susana; Megías, Zoraida; Martínez, Cecilia; García, Alicia; Aguado, Encarnación; Chileh, Tarik; López-Alonso, Diego; García-Maroto, Federico; Kejnovský, Eduard; Široký, Jiří; Kubát, Zdeněk; Králová, Tereza; Vyskot, Boris; Jamilena, Manuel

2017-01-01

Sex determination in Rumex acetosa, a dioecious plant with a complex XY 1 Y 2 sex chromosome system (females are XX and males are XY 1 Y 2 ), is not controlled by an active Y chromosome but depends on the ratio between the number of X chromosomes and autosomes. To gain insight into the molecular mechanisms of sex determination, we generated a subtracted cDNA library enriched in genes specifically or predominantly expressed in female floral buds in early stages of development, when sex determination mechanisms come into play. In the present paper, we report the molecular and functional characterization of FEM32, a gene encoding a protein that shares a common architecture with proteins in different plants, animals, bacteria and fungi of the aerolysin superfamily; many of these function as β pore-forming toxins. The expression analysis, assessed by northern blot, RT-PCR and in situ hybridization, demonstrates that this gene is specifically expressed in flowers in both early and late stages of development, although its transcripts accumulate much more in female flowers than in male flowers. The ectopic expression of FEM32 under both the constitutive promoter 35S and the flower-specific promoter AP3 in transgenic tobacco showed no obvious alteration in vegetative development but was able to alter floral organ growth and pollen fertility. The 35S::FEM32 and AP3::FEM32 transgenic lines showed a reduction in stamen development and pollen viability, as well as a diminution in fruit set, fruit development and seed production. Compared with other floral organs, pistil development was, however, enhanced in plants overexpressing FEM32. According to these effects, it is likely that FEM32 functions in Rumex by arresting stamen and pollen development during female flower development. The aerolysin-like pore-forming proteins of eukaryotes are mainly involved in defence mechanisms against bacteria, fungi and insects and are also involved in apoptosis and programmed cell death (PCD

Overexpression of Rad in muscle worsens diet-induced insulin resistance and glucose intolerance and lowers plasma triglyceride level

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Ilany, Jacob; Bilan, Philip J.; Kapur, Sonia; Caldwell, James S.; Patti, Mary-Elizabeth; Marette, Andre; Kahn, C. Ronald

2006-03-01

Rad is a low molecular weight GTPase that is overexpressed in skeletal muscle of some patients with type 2 diabetes mellitus and/or obesity. Overexpression of Rad in adipocytes and muscle cells in culture results in diminished insulin-stimulated glucose uptake. To further elucidate the potential role of Rad in vivo, we have generated transgenic (tg) mice that overexpress Rad in muscle using the muscle creatine kinase (MCK) promoter-enhancer. Rad tg mice have a 6- to 12-fold increase in Rad expression in muscle as compared to wild-type littermates. Rad tg mice grow normally and have normal glucose tolerance and insulin sensitivity, but have reduced plasma triglyceride levels. On a high-fat diet, Rad tg mice develop more severe glucose intolerance than the wild-type mice; this is due to increased insulin resistance in muscle, as exemplified by a rightward shift in the dose-response curve for insulin stimulated 2-deoxyglucose uptake. There is also a unexpected further reduction of the plasma triglyceride levels that is associated with increased levels of lipoprotein lipase in the Rad tg mice. These results demonstrate a potential synergistic interaction between increased expression of Rad and high-fat diet in creation of insulin resistance and altered lipid metabolism present in type 2 diabetes. diabetes mellitus | glucose transport | RGK GTPase | transgenic mouse

Overexpression of osteoprotegerin promotes preosteoblast differentiation to mature osteoblasts

NARCIS (Netherlands)

Yu, Hongyou; de Vos, Paul; Ren, Yijin

OBJECTIVE: The hypothesis of the present study is that overexpression of osteoprotegerin (OPG) promotes preosteoblast maturation. MATERIALS AND METHODS: The preosteoblast cell line MC3T3-E1 was transfected with OPG overexpression. OPG expression was confirmed by enzyme-linked immunosorbent assay

Immunocytochemical Localization of Calbindin D28K, Calretinin, and Parvalbumin in Bat Superior Colliculus

International Nuclear Information System (INIS)

Jeong, Se-Jin; Kim, Hyun-Ho; Lee, Won-Sig; Jeon, Chang-Jin

2014-01-01

The purpose of this study was to investigate the localization of cells containing the calcium-binding proteins (CBPs) calbindin D28K (CB), calretinin (CR), and parvalbumin (PV) in the superior colliculus (SC) of the bat using immunocytochemistry. CB-immunoreactive (IR) cells formed a laminar tier within the upper superficial gray layer (SGL), while CR-IR cells were widely distributed within the optic layer (OL). Scattered CR-IR cells were also found within the intermediate gray, white, and deep gray layers. By contrast, PV-IR cells formed a laminar tier within the lower SGL and upper OL. Scattered PV-IR cells were also found throughout the intermediate layers, but without a specific laminar pattern. The CBP-IR cells varied in size and morphology: While most of the CB-IR cells in the superficial layers were small round or oval cells, most CR-IR cells in the intermediate and deep layers were large stellate cells. By contrast, PV-IR cells were small to large in size and included round or oval, stellate, vertical fusiform, and horizontal cells. The average diameters of the CB-, CR-, and PV-IR cells were 11.59, 17.17, and 12.60 μm, respectively. Double-immunofluorescence revealed that the percentage of co-localization with GABA-IR cells was 0.0, 0.0, and 10.27% of CB-, CR-, and PV-IR cells, respectively. These results indicate that CBP distribution patterns in the bat SC are unique compared with other mammalian SCs, which suggest functional diversity of these proteins in visually guided behaviors

Overexpression of ZmIRT1 and ZmZIP3 Enhances Iron and Zinc Accumulation in Transgenic Arabidopsis.

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Suzhen Li

Full Text Available Iron and zinc are important micronutrients for both the growth and nutrient availability of crop plants, and their absorption is tightly controlled by a metal uptake system. Zinc-regulated transporters, iron-regulated transporter-like proteins (ZIP, is considered an essential metal transporter for the acquisition of Fe and Zn in graminaceous plants. Several ZIPs have been identified in maize, although their physiological function remains unclear. In this report, ZmIRT1 was shown to be specifically expressed in silk and embryo, whereas ZmZIP3 was a leaf-specific gene. Both ZmIRT1 and ZmZIP3 were shown to be localized to the plasma membrane and endoplasmic reticulum. In addition, transgenic Arabidopsis plants overexpressing ZmIRT1 or ZmZIP3 were generated, and the metal contents in various tissues of transgenic and wild-type plants were examined based on ICP-OES and Zinpyr-1 staining. The Fe and Zn concentration increased in roots and seeds of ZmIRT1-overexpressing plants, while the Fe content in shoots decreased. Overexpressing ZmZIP3 enhanced Zn accumulation in the roots of transgenic plants, while that in shoots was repressed. In addition, the transgenic plants showed altered tolerance to various Fe and Zn conditions compared with wild-type plants. Furthermore, the genes associated with metal uptake were stimulated in ZmIRT1 transgenic plants, while those involved in intra- and inter- cellular translocation were suppressed. In conclusion, ZmIRT1 and ZmZIP3 are functional metal transporters with different ion selectivities. Ectopic overexpression of ZmIRT1 may stimulate endogenous Fe uptake mechanisms, which may facilitate metal uptake and homeostasis. Our results increase our understanding of the functions of ZIP family transporters in maize.

Genetic alterations in head and neck squamous cell carcinomas

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Nagai M.A.

1999-01-01

Full Text Available The genetic alterations observed in head and neck cancer are mainly due to oncogene activation (gain of function mutations and tumor suppressor gene inactivation (loss of function mutations, leading to deregulation of cell proliferation and death. These genetic alterations include gene amplification and overexpression of oncogenes such as myc, erbB-2, EGFR and cyclinD1 and mutations, deletions and hypermethylation leading to p16 and TP53 tumor suppressor gene inactivation. In addition, loss of heterozygosity in several chromosomal regions is frequently observed, suggesting that other tumor suppressor genes not yet identified could be involved in the tumorigenic process of head and neck cancers. The exact temporal sequence of the genetic alterations during head and neck squamous cell carcinoma (HNSCC development and progression has not yet been defined and their diagnostic or prognostic significance is controversial. Advances in the understanding of the molecular basis of head and neck cancer should help in the identification of new markers that could be used for the diagnosis, prognosis and treatment of the disease.

FOX-2 Dependent Splicing of Ataxin-2 Transcript Is Affected by Ataxin-1 Overexpression

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Welzel, Franziska; Kaehler, Christian; Isau, Melanie; Hallen, Linda; Lehrach, Hans; Krobitsch, Sylvia

2012-01-01

Alternative splicing is a fundamental posttranscriptional mechanism for controlling gene expression, and splicing defects have been linked to various human disorders. The splicing factor FOX-2 is part of a main protein interaction hub in a network related to human inherited ataxias, however, its impact remains to be elucidated. Here, we focused on the reported interaction between FOX-2 and ataxin-1, the disease-causing protein in spinocerebellar ataxia type 1. In this line, we further evaluated this interaction by yeast-2-hybrid analyses and co-immunoprecipitation experiments in mammalian cells. Interestingly, we discovered that FOX-2 localization and splicing activity is affected in the presence of nuclear ataxin-1 inclusions. Moreover, we observed that FOX-2 directly interacts with ataxin-2, a protein modulating spinocerebellar ataxia type 1 pathogenesis. Finally, we provide evidence that splicing of pre-mRNA of ataxin-2 depends on FOX-2 activity, since reduction of FOX-2 levels led to increased skipping of exon 18 in ataxin-2 transcripts. Most striking, we observed that ataxin-1 overexpression has an effect on this splicing event as well. Thus, our results demonstrate that FOX-2 is involved in splicing of ataxin-2 transcripts and that this splicing event is altered by overexpression of ataxin-1. PMID:22666429

Overexpression of SIRT1 in mouse forebrain impairs lipid/glucose metabolism and motor function.

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Dongmei Wu

Full Text Available SIRT1 plays crucial roles in glucose and lipid metabolism, and has various functions in different tissues including brain. The brain-specific SIRT1 knockout mice display defects in somatotropic signaling, memory and synaptic plasticity. And the female mice without SIRT1 in POMC neuron are more sensitive to diet-induced obesity. Here we created transgenic mice overexpressing SIRT1 in striatum and hippocampus under the control of CaMKIIα promoter. These mice, especially females, exhibited increased fat accumulation accompanied by significant upregulation of adipogenic genes in white adipose tissue. Glucose tolerance of the mice was also impaired with decreased Glut4 mRNA levels in muscle. Moreover, the SIRT1 overexpressing mice showed decreased energy expenditure, and concomitantly mitochondria-related genes were decreased in muscle. In addition, these mice showed unusual spontaneous physical activity pattern, decreased activity in open field and rotarod performance. Further studies demonstrated that SIRT1 deacetylated IRS-2, and upregulated phosphorylation level of IRS-2 and ERK1/2 in striatum. Meanwhile, the neurotransmitter signaling in striatum and the expression of endocrine hormones in hypothalamus and serum T3, T4 levels were altered. Taken together, our findings demonstrate that SIRT1 in forebrain regulates lipid/glucose metabolism and motor function.

Cytogenetic evaluation of human glial tumors: correlation of overexpression of epidermal growth factor receptor (EGFB) with abnormalities of chromosome 7

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Bell, C.W.

1987-01-01

Chromosome banding analysis of human glial tumors were performed using G- and Q-banding techniques in an attempt to establish recurring sites of chromosome change. Results revealed a nonrandom karyotypic profile including aneuploidy and considerable variation in chromosome number (range 40 → 200). All tumors examined displayed numerical abnormalities, with the most common numeric change being a gain of chromosome 7. An attempt was then made to correlate the observed chromosome 7 changes with activation of the cellular proto-oncogene c-erb-B, whose produce is the epidermal growth factor receptor (EGFR). Six human glial tumors were analyzed for 125 I-EGF binding, EGFR gene copy number, EGFR gene rearrangement, mRNA expression, and karyotypic profile. Saturation analysis at 4 0 C revealed significant numbers of EGFR's in all 6 tumors. Southern blotting analysis utilizing cDNA probes for the EGFR failed to demonstrate significant amplification or structural rearrangement of the EFGR gene. The results suggest that overexpression of the EGFR may be related to an alternative mechanism, other than gene amplification and elevated mRNA levels, such as the regulation of receptor biosynthesis and degradation. In summary, findings indicate that alterations of chromosome 7 are the most prevalent chromosomal change in human glial tumors, and that these alterations may lead to overexpression of the protooncogene c-erb-B

In vivo overexpression of Emi1 promotes chromosome instability and tumorigenesis.

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Vaidyanathan, S; Cato, K; Tang, L; Pavey, S; Haass, N K; Gabrielli, B G; Duijf, P H G

2016-10-13

Cell cycle genes are often aberrantly expressed in cancer, but how their misexpression drives tumorigenesis mostly remains unclear. From S phase to early mitosis, EMI1 (also known as FBXO5) inhibits the anaphase-promoting complex/cyclosome, which controls cell cycle progression through the sequential degradation of various substrates. By analyzing 7403 human tumor samples, we find that EMI1 overexpression is widespread in solid tumors but not in blood cancers. In solid cancers, EMI1 overexpression is a strong prognostic marker for poor patient outcome. To investigate causality, we generated a transgenic mouse model in which we overexpressed Emi1. Emi1-overexpressing animals develop a wide variety of solid tumors, in particular adenomas and carcinomas with inflammation and lymphocyte infiltration, but not blood cancers. These tumors are significantly larger and more penetrant, abundant, proliferative and metastatic than control tumors. In addition, they are highly aneuploid with tumor cells frequently being in early mitosis and showing mitotic abnormalities, including lagging and incorrectly segregating chromosomes. We further demonstrate in vitro that even though EMI1 overexpression may cause mitotic arrest and cell death, it also promotes chromosome instability (CIN) following delayed chromosome alignment and anaphase onset. In human solid tumors, EMI1 is co-expressed with many markers for CIN and EMI1 overexpression is a stronger marker for CIN than most well-established ones. The fact that Emi1 overexpression promotes CIN and the formation of solid cancers in vivo indicates that Emi1 overexpression actively drives solid tumorigenesis. These novel mechanistic insights have important clinical implications.

ABF2, an ABRE-binding bZIP factor, is an essential component of glucose signaling and its overexpression affects multiple stress tolerance.

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Kim, Sunmi; Kang, Jung-Youn; Cho, Dong-Im; Park, Ji Hye; Kim, Soo Young

2004-10-01

Phytohormone abscisic acid (ABA) regulates stress-responsive gene expression during vegetative growth, which is mediated largely by cis-elements sharing the ACGTGGC consensus. Although many transcription factors are known to bind the elements in vitro, only a few have been demonstrated to have in vivo functions and their specific roles in ABA/stress responses are mostly unknown. Here, we report that ABF2, an ABF subfamily member of bZIP proteins interacting with the ABA-responsive elements, is involved in ABA/stress responses. Its overexpression altered ABA sensitivity, dehydration tolerance, and the expression levels of ABA/stress-regulated genes. Furthermore, ABF2 overexpression promoted glucose-induced inhibition of seedling development, whereas its mutation impaired glucose response. The reduced sugar sensitivity was not observed with mutants of two other ABF family members, ABF3 and ABF4. Instead, these mutants displayed defects in ABA, salt, and dehydration responses, which were not observed with the abf2 mutant. Our data indicate distinct roles of ABF family members: whereas ABF3 and ABF4 play essential roles in ABA/stress responses, ABF2 is required for normal glucose response. We also show that ABF2 overexpression affects multiple stress tolerance.

Epidermal growth factor receptor structural alterations in gastric cancer

International Nuclear Information System (INIS)

Moutinho, Cátia; Mateus, Ana R; Milanezi, Fernanda; Carneiro, Fátima; Seruca, Raquel; Suriano, Gianpaolo

2008-01-01

EGFR overexpression has been described in many human tumours including gastric cancer. In NSCLC patients somatic EGFR mutations, within the kinase domain of the protein, as well as gene amplification were associated with a good clinical response to EGFR inhibitors. In gastric tumours data concerning structural alterations of EGFR remains controversial. Given its possible therapeutic relevance, we aimed to determine the frequency and type of structural alterations of the EGFR gene in a series of primary gastric carcinomas. Direct sequencing of the kinase domain of the EGFR gene was performed in a series of 77 primary gastric carcinomas. FISH analysis was performed in 30 cases. Association studies between EGFR alterations and the clinical pathological features of the tumours were performed. Within the 77 primary gastric carcinomas we found two EGFR somatic mutations and several EGFR polymorphisms in exon 20. Six different intronic sequence variants of EGFR were also found. Four gastric carcinomas showed balanced polysomy or EGFR gene amplification. We verified that gastric carcinoma with alterations of EGFR (somatic mutations or copy number variation) showed a significant increase of tumour size (p = 0.0094) in comparison to wild-type EGFR carcinomas. We demonstrate that EGFR structural alterations are rare in gastric carcinoma, but whenever present, it leads to tumour growth. We considered that searching for EGFR alterations in gastric cancer is likely to be clinically important in order to identify patients susceptible to respond to tyrosine kinase inhibitors

HER2 amplification, overexpression and score criteria in esophageal adenocarcinoma

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Hu, Yingchuan; Bandla, Santhoshi; Godfrey, Tony E.; Tan, Dongfeng; Luketich, James D.; Pennathur, Arjun; Qiu, Xing; Hicks, David G.; Peters, Jeffrey; Zhou, Zhongren

2011-01-01

The HER2 oncogene was recently reported to be amplified and overexpressed in esophageal adenocarcinoma. However, the relationship of HER2 amplification in esophageal adenocarcinoma with prognosis has not been well defined. The scoring systems for clinically evaluating HER2 in esophageal adenocarcinoma are not established. The aims of the study were to establish a HER2 scoring system and comprehensively investigate HER2 amplification and overexpression in esophageal adenocarcinoma and its precursor lesion. Using a tissue microarray, containing 116 cases of esophageal adenocarcinoma, 34 cases of BE, 18 cases of low grade dysplasia and 15 cases of high grade dysplasia, HER2 amplification and overexpression were analyzed by HercepTest and CISH methods. The amplification frequency in an independent series of 116 esophageal adenocarcinoma samples was also analyzed using Affymetrix SNP 6.0 microarrays. In our studies, we have found that HER2 amplification does not associate with poor prognosis in total 232 esophageal adenocarcinoma patients by CISH and high density microarrays. We further confirm the similar frequency of HER2 amplification by CISH (18.10%; 21/116) and SNP 6.0 microarrays (16.4%, 19/116) in esophageal adenocarcinoma. HER2 protein overexpression was observed in 12.1 % (14/116) of esophageal adenocarcinoma and 6.67% (1/15) of HGD. No HER2 amplification or overexpression was identified in BE or LGD. All HER2 protein overexpression cases showed HER2 gene amplification. Gene amplification was found to be more frequent by CISH than protein overexpression in esophageal adenocarcinoma (18.10% vs 12.9%). A modified two-step model for esophageal adenocarcinoma HER-2 testing is recommend for clinical esophageal adenocarcinoma HER-2 trial. PMID:21460800

Concurrent nuclear ERG and MYC protein overexpression defines a subset of locally advanced prostate cancer: Potential opportunities for synergistic targeted therapeutics.

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Udager, Aaron M; DeMarzo, Angelo M; Shi, Yang; Hicks, Jessica L; Cao, Xuhong; Siddiqui, Javed; Jiang, Hui; Chinnaiyan, Arul M; Mehra, Rohit

2016-06-01

Recurrent ERG gene fusions, the most common genetic alterations in prostate cancer, drive overexpression of the nuclear transcription factor ERG, and are early clonal events in prostate cancer progression. The nuclear transcription factor MYC is also frequently overexpressed in prostate cancer and may play a role in tumor initiation and/or progression. The relationship between nuclear ERG and MYC protein overexpression in prostate cancer, as well as the clinicopathologic characteristics and prognosis of ERG-positive/MYC high tumors, is not well understood. Immunohistochemistry (IHC) for ERG and MYC was performed on formalin-fixed, paraffin-embedded tissue from prostate cancer tissue microarrays (TMAs), and nuclear staining was scored semi-quantitatively (IHC product score range = 0-300). Correlation between nuclear ERG and MYC protein expression and association with clinicopathologic parameters and biochemical recurrence after radical prostatectomy was assessed. 29.1% of all tumor nodules showed concurrent nuclear ERG and MYC protein overexpression (i.e., ERG-positive/MYC high), including 35.0% of secondary nodules. Overall, there was weak positive correlation between ERG and MYC expression across all tumor nodules (rpb  = 0.149, P = 0.045), although this correlation was strongest in secondary nodules (rpb  = 0.520, P = 0.019). In radical prostatectomy specimens, ERG-positive/MYC high tumors were positively associated with the presence of extraprostatic extension (EPE), relative to all other ERG/MYC expression subgroups, however, there was no significant association between concurrent nuclear ERG and MYC protein overexpression and time to biochemical recurrence. Concurrent nuclear ERG and MYC protein overexpression is common in prostate cancer and defines a subset of locally advanced tumors. Recent data indicates that BET bromodomain proteins regulate ERG gene fusion and MYC gene expression in prostate cancer, suggesting possible synergistic

Prognostic implication of NQO1 overexpression in hepatocellular carcinoma.

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Lin, Lijuan; Sun, Jie; Tan, Yan; Li, Zhenling; Kong, Fanyong; Shen, Yue; Liu, Chao; Chen, Litian

2017-11-01

To explore the role of NQO1 overexpression for prognostic implication in hepatocellular carcinoma (HCC), NQO1 mRNA levels were detected in HCC fresh tissue samples of HCC and nontumor tissues, respectively. One hundred fifty-six cases of HCC meeting strict follow-up criteria were selected for immunohistochemical staining of NQO1 protein. Correlations between NQO1 overexpression and clinicopathological features of HCC were evaluated using χ 2 tests, survival rates were calculated using the Kaplan-Meier method, and the relationship between prognostic factors and patient 5-year survival was analyzed using Cox proportional hazards analysis. In results, the levels of NQO1 mRNA were significantly up-regulated in 14 fresh tissue samples of HCC. Immunohistochemical analysis showed that the NQO1 expression and overexpression rates were significantly higher in HCC samples compared with either adjacent nontumor tissues or normal liver tissues. NQO1 overexpression correlated to tumor size, venous infiltration and late pTNM stage of HCC. NQO1 overexpression was also related to low disease-free survival and 5-year survival rates. In the late-stage group, disease-free and 5-year survival rates of patients with NQO1 overexpression were significantly lower than those of patients without NQO1 expression. Further analysis using a Cox proportional hazards regression model revealed that NQO1 expression emerged as a significant independent hazard factor for the 5-year survival rate of patients with HCC. Therefore, NQO1 plays an important role in the progression of HCC. NQO1 may potentially be used as an independent biomarker for prognostic evaluation of HCC. Copyright © 2017. Published by Elsevier Inc.

Tobacco, alcohol, and p53 overexpression in early colorectal neoplasia

International Nuclear Information System (INIS)

Terry, Mary Beth; Neugut, Alfred I; Mansukhani, Mahesh; Waye, Jerome; Harpaz, Noam; Hibshoosh, Hanina

2003-01-01

The p53 tumor suppressor gene is commonly mutated in colorectal cancer. While the effect of p53 mutations on colorectal cancer prognosis has been heavily studied, less is known about how epidemiologic risk factors relate to p53 status, particularly in early colorectal neoplasia prior to clinically invasive colorectal cancer (including adenomas, carcinoma in situ (CIS), and intramucosal carcinoma). We examined p53 status, as measured by protein overexpression, in 157 cases with early colorectal neoplasia selected from three New York City colonoscopy clinics. After collecting paraffin-embedded tissue blocks, immunohistochemistry was performed using an anti-p53 monoclonal mouse IgG 2 a [BP53-12-1] antibody. We analyzed whether p53 status was different for risk factors for colorectal neoplasia relative to a polyp-free control group (n = 508). p53 overexpression was found in 10.3%, 21.7%, and 34.9%, of adenomatous polyps, CIS, and intramucosal cases, respectively. Over 90% of the tumors with p53 overexpression were located in the distal colon and rectum. Heavy cigarette smoking (30+ years) was associated with cases not overexpressing p53 (OR = 1.8, 95% CI = 1.1–2.9) but not with those cases overexpressing p53 (OR = 1.0, 95% CI = 0.4–2.6). Heavy beer consumption (8+ bottles per week) was associated with cases overexpressing p53 (OR = 4.0, 95% CI = 1.3–12.0) but not with cases without p53 overexpression (OR = 1.6, 95% CI = 0.7–3.7). Our findings that p53 overexpression in early colorectal neoplasia may be positively associated with alcohol intake and inversely associated with cigarette smoking are consistent with those of several studies of p53 expression and invasive cancer, and suggest that there may be relationships of smoking and alcohol with p53 early in the adenoma to carcinoma sequence

Hypermethylation of the 5′ CpG island of the p14ARF flanking exon 1β in human colorectal cancer displaying a restricted pattern of p53 overexpression concomitant with increased MDM2 expression

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Nyiraneza Christine

2012-06-01

Full Text Available Abstract Background It has been suggested that inactivation of p14ARF, a tumor suppressor central to regulating p53 protein stability through interaction with the MDM2 oncoprotein, abrogates p53 activity in human tumors retaining the wild-type TP53 gene. Differences in expression of tumor suppressor genes are frequently associated with cancer. We previously reported on a pattern of restricted p53 immunohistochemical overexpression significantly associated with microsatellite instability (MSI, low TP53 mutation frequency, and MDM2 overexpression in colorectal cancers (CRCs. In this study, we investigated whether p14ARF alterations could be a mechanism for disabling the p53 pathway in this subgroup of CRCs. Results Detailed maps of the alterations in the p14ARF gene were determined in a cohort of 98 CRCs to detect both nucleotide and copy-number changes. Methylation-specific PCR combined with bisulfite sequencing was used to evaluate the prevalence and distribution of p14ARF methylation. p14ARF alterations were then correlated with MSI status, TP53 mutations, and immunohistochemical expression of p53 and MDM2. The frequency of p14ARF mutations was extremely low (1/98; 1%, whereas coexistence of methylated and unmethylated alleles in both tumors and normal colon mucosa was common (91/98; 93%. Only seven of ninety-eight tumors (7% had a distinct pattern of methylation compared with normal colon mucosa. Evaluation of the prevalence and distribution of p14ARF promoter methylation in a region containing 27 CpG sites in 35 patients showed a range of methylated CpG sites in tumors (0 to 25 (95% CI 1 to 13 versus 0 to 17 (95% CI 0 to 2 in adjacent colon mucosa (P = 0.004. Hypermethylation of the p14ARF promoter was significantly correlated with the restricted p53 overexpression pattern (P = 0.03, and MDM2 overexpression (P = 0.02, independently of MSI phenotype. Although no significant correlation between p14ARF methylation and TP53 mutational

FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1.

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Sandra J Feeney

Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

CENPA overexpression promotes genome instability in pRb-depleted human cells

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Lentini Laura

2009-12-01

Full Text Available Abstract Background Aneuploidy is a hallmark of most human cancers that arises as a consequence of chromosomal instability and it is frequently associated with centrosome amplification. Functional inactivation of the Retinoblastoma protein (pRb has been indicated as a cause promoting chromosomal instability as well centrosome amplification. However, the underlying molecular mechanism still remains to be clarified. Results Here we show that pRb depletion both in wild type and p53 knockout HCT116 cells was associated with the presence of multipolar spindles, anaphase bridges, lagging chromosomes and micronuclei harbouring whole chromosomes. In addition aneuploidy caused by pRb acute loss was not affected by p53 loss. Quantitative real-time RT-PCR showed that pRB depletion altered expression of genes involved in centrosome duplication, kinetochore assembly and in the Spindle Assembly Checkpoint (SAC. However, despite MAD2 up-regulation pRb-depleted cells seemed to have a functional SAC since they arrested in mitosis after treatments with mitotic poisons. Moreover pRb-depleted HCT116 cells showed BRCA1 overexpression that seemed responsible for MAD2 up-regulation. Post-transcriptional silencing of CENPA by RNA interference, resulting in CENP-A protein levels similar to those present in control cells greatly reduced aneuploid cell numbers in pRb-depleted cells. Conclusion Altogether our findings indicate a novel aspect of pRb acute loss that promotes aneuploidy mainly by inducing CENPA overexpression that in turn might induce micronuclei by affecting the correct attachment of spindle microtubules to kinetochores.

Senescence-Induced Alterations of Laminin Chain Expression Modulate Tumorigenicity of Prostate Cancer Cells

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Cynthia C.T. Sprenger

2008-12-01

Full Text Available Prostate cancer is an age-associated epithelial cancer, and as such, it contributes significantly to the mortality of the elderly. Senescence is one possible mechanism by which the body defends itself against various epithelial cancers. Senescent cells alter the microenvironment, in part, through changes to the extracellular matrix. Laminins (LMs are extracellular proteins important to both the structure and function of the microenvironment. Overexpression of the senescence-associated gene mac25 in human prostate cancer cells resulted in increased mRNA levels of the LM α4 and β2 chains compared to empty vector control cells. The purpose of this study was to examine the effects of these senescence-induced LM chains on tumorigenicity of prostate cancer cells. We created stable M12 human prostate cancer lines overexpressing either the LM α4 or β2 chain or both chains. Increased expression of either the LM α4 or β2 chain resulted in increased in vitro migration and in vivo tumorigenicity of those cells, whereas high expression of both chains led to decreased in vitro proliferation and in vivo tumorigenicity compared to M12 control cells. This study demonstrates that senescent prostate epithelial cells can alter the microenvironment and that these changes modulate progression of prostate cancer.

Hippocampal neuroligin-2 overexpression leads to reduced aggression and inhibited novelty reactivity in rats.

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Christine Kohl

Full Text Available Disturbances of the excitation/inhibition (E/I balance in the brain were recently suggested as potential factors underlying disorders like autism and schizophrenia resulting in associated behavioral alterations including changes in social and emotional behavior as well as abnormal aggression. Neuronal cell adhesion molecules (nCAMs and mutations in these genes were found to be strongly implicated in the pathophysiology of these disorders. Neuroligin2 (nlgn2 is a postsynaptic cell adhesion molecule, which is predominantly expressed at inhibitory synapses and required for synapse specification and stabilization. Changes in the expression of nlgn2 were shown to result in alterations of social behavior as well as altered inhibitory synaptic transmission, hence modifying the E/I balance. In our study, we focused on the role of nlgn2 in the dorsal hippocampus in the regulation of emotional and social behaviors. To this purpose, we injected an AAV construct overexpressing nlgn2 in the hippocampus of rats and investigated the effects on behavior and on markers for the E/I ratio. We could show an increase in GAD65, a GABA-synthesizing protein in neuronal terminals, and furthermore, reduced exploration of novel stimuli and less offensive behavior. Our data suggest nlgn2 in the hippocampus to be strongly implicated in maintaining the E/I balance in the brain and thereby modulating social and emotional behavior.

The frequency and clinical impact of HER2 alterations in lung adenocarcinoma.

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Eun Kyung Kim

Full Text Available Human epidermal growth factor receptor 2 (HER2 or ErbB2 can be overexpressed, amplified and/or mutated in malignant tumors, and is a candidate for therapeutic targeting. However, molecular associations and clinical significances of these alterations were controversial in lung cancer. In this study, we investigated the frequency and clinicopathological significance of HER2 dysregulation in patients with lung adenocarcinoma. HER2 protein overexpression, gene amplification, and gene mutation were evaluated by immunohistochemistry (IHC, silver in situ hybridization, and direct sequencing, respectively. The H-scoring method and American Society of Clinical Oncology/College of American Pathologists breast cancer guidelines were used to interpret IHC results. Genetic analyses of EGFR and KRAS mutations, and of ALK and ROS1 rearrangements, were also performed. Of the 321 adenocarcinoma patients identified, HER2 overexpression (H-score ≥200 and gene amplification were found in 6 (1.9% and 46 (14.3%, respectively. HER2 overexpression was correlated with papillary predominant histology; furthermore, it indicated poor overall survival and was an independent prognostic factor. HER2 amplification was associated with pleural invasion and showed a tendency towards shorter overall and disease-free survival. High-level gene amplification (HER2/CEP17 ratio ≥5 or copy number ≥10 was a poor prognostic factor for disease-free survival. HER2 mutations were detected in 6.7% (7 of 104 of driver oncogene-negative adenocarcinomas. Our study suggests that HER2 overexpression or amplification is a poor prognostic factor in lung adenocarcinoma, although the frequency of such events is low. Since molecular targeted agents are being tested in clinical trials, awareness of the specific HER2 status can influence the prognostic stratification and treatment of patients with molecularly defined subsets of lung adenocarcinoma.

Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells

DEFF Research Database (Denmark)

Honjo, Y; Hrycyna, C A; Yan, Q W

2001-01-01

A disparity was noted in the transport of rhodamine 123 among nine MXR/BCRP/ABCP-overexpressing cells studied; all demonstrated mitoxantrone transport, whereas only two effluxed rhodamine 123. When the MXR/BCRP/ABCP gene was sequenced in the cell lines studied, differences were noted at amino acid...... 482, predicted to be at the start of the third transmembrane domain. Sequencing genomic DNA revealed wild-type MXR/BCRP/ABCP to have an arginine at position 482. Cells having a threonine or glycine at position 482 were able to efflux rhodamine 123, whereas cells having an arginine were not. A vaccinia...... virus expression system confirmed that rhodamine as well as doxorubicin efflux is observed with R482T or R482G but not with the wild-type R482; all three MXR/BCRP/ABCP forms transported mitoxantrone. Cross-resistance studies suggest that, compared with wild-type MXR/BCRP/ABCP, cells having an R482T...

Reduced grain chalkiness and its possible physiological mechanism in transgenic rice overexpressing l-GalLDH

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Le Yu

2015-04-01

Full Text Available Chalkiness is one of the key factors determining rice quality and price. Ascorbic acid (Asc is a major plant antioxidant that performs many functions in plants. l-Galactono-1,4-lactone dehydrogenase (l-GalLDH, EC1.3.2.3 is an enzyme that catalyzes the final step of Asc biosynthesis in plants. Here we show that the l-GalLDH-overexpressing transgenic rice, GO-2, which has constitutively higher leaf Asc content than wild-type (WT plants, exhibits significantly reduced grain chalkiness. Higher foliar ascorbate/dehydroascorbate (Asc/DHA ratios at 40, 60, 80, and 100 days of plant age were observed in GO-2. Further investigation showed that the enhanced level of Asc resulted in a significantly higher ribulose-1,5-bisphosphate (RuBP carboxylase/oxygenase (Rubisco protein level in GO-2 at 80 days. In addition, levels of abscisic acid (ABA and jasmonic acid (JA were lower in GO-2 at 60, 80, and 100 days. The results we present here indicate that the enhanced level of Asc is likely responsible for changing redox homeostasis in key developmental stages associated with grain filling and alters grain chalkiness in the l-GalLDH-overexpressing transgenic by maintaining photosynthetic function and affecting phytohormones associated with grain filling.

Claudin-4 Overexpression in Epithelial Ovarian Cancer Is Associated with Hypomethylation and Is a Potential Target for Modulation of Tight Junction Barrier Function Using a C-Terminal Fragment of Clostridium perfringens Enterotoxin

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Babak Litkouhi

2007-04-01

Full Text Available BACKGROUND: Claudin-4, a tight junction (TJ protein and receptor for the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE, is overexpressed in epithelial ovarian cancer (EOC. Previous research suggests DNA methylation is a mechanism for claudin-4 overexpression in cancer and that C-CPE acts as an absorption-enhancing agent in claudin-4expressing cells. We sought to correlate claudin-4 overexpression in EOC with clinical outcomes and TJ barrier function, investigate DNA methylation as a mechanism for overexpression, and evaluate the effect of C-CPE on the TJ. METHODS: Claudin-4 expression in EOC was quantified and correlated with clinical outcomes. Claudin-4 methylation status was determined, and claudin-4-negative cell lines were treated with a demethylating agent. Electric cell-substrate impedance sensing was used to calculate junctional (paracellular resistance (Rb in EOC cells after claudin-4 silencing and after C-CPE treatment. RESULTS: Claudin4 overexpression in EOC does not correlate with survival or other clinical endpoints and is associated with hypomethylation. Claudin-4 overexpression correlates with Rb and C-CPE treatment of EOC cells significantly decreased Rb in a dose- and claudin-4-dependent noncytotoxic manner. CONCLUSIONS: C-CPE treatment of EOC cells leads to altered TJ function. Further research is needed to determine the potential clinical applications of C-CPE in EOC drug delivery strategies.

Transgenic overexpression of 14-3-3 zeta protects hippocampus against endoplasmic reticulum stress and status epilepticus in vivo.

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Gary P Brennan

Full Text Available 14-3-3 proteins are ubiquitous molecular chaperones that are abundantly expressed in the brain where they regulate cell functions including metabolism, the cell cycle and apoptosis. Brain levels of several 14-3-3 isoforms are altered in diseases of the nervous system, including epilepsy. The 14-3-3 zeta (ζ isoform has been linked to endoplasmic reticulum (ER function in neurons, with reduced levels provoking ER stress and increasing vulnerability to excitotoxic injury. Here we report that transgenic overexpression of 14-3-3ζ in mice results in selective changes to the unfolded protein response pathway in the hippocampus, including down-regulation of glucose-regulated proteins 78 and 94, activating transcription factors 4 and 6, and Xbp1 splicing. No differences were found between wild-type mice and transgenic mice for levels of other 14-3-3 isoforms or various other 14-3-3 binding proteins. 14-3-3ζ overexpressing mice were potently protected against cell death caused by intracerebroventricular injection of the ER stressor tunicamycin. 14-3-3ζ overexpressing mice were also potently protected against neuronal death caused by prolonged seizures. These studies demonstrate that increased 14-3-3ζ levels protect against ER stress and seizure-damage despite down-regulation of the unfolded protein response. Delivery of 14-3-3ζ may protect against pathologic changes resulting from prolonged or repeated seizures or where injuries provoke ER stress.

Docosahexaenoic Acid Modulates a HER2-Associated Lipogenic Phenotype, Induces Apoptosis, and Increases Trastuzumab Action in HER2-Overexpressing Breast Carcinoma Cells.

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Ravacci, Graziela Rosa; Brentani, Maria Mitzi; Tortelli, Tharcisio Citrângulo; Torrinhas, Raquel Suzana M M; Santos, Jéssica Reis; Logullo, Angela Flávia; Waitzberg, Dan Linetzky

2015-01-01

In breast cancer, lipid metabolic alterations have been recognized as potential oncogenic stimuli that may promote malignancy. To investigate whether the oncogenic nature of lipogenesis closely depends on the overexpression of HER2 protooncogene, the normal breast cell line, HB4a, was transfected with HER2 cDNA to obtain HER2-overexpressing HB4aC5.2 cells. Both cell lines were treated with trastuzumab and docosahexaenoic acid. HER2 overexpression was accompanied by an increase in the expression of lipogenic genes involved in uptake (CD36), transport (FABP4), and storage (DGAT) of exogenous fatty acids (FA), as well as increased activation of "de novo" FA synthesis (FASN). We further investigate whether this lipogenesis reprogramming might be regulated by mTOR/PPARγ pathway. Inhibition of the mTORC1 pathway markers, p70S6 K1, SREBP1, and LIPIN1, as well as an increase in DEPTOR expression (the main inhibitor of the mTOR) was detected in HB4aC5.2. Based on these results, a PPARγ selective antagonist, GW9662, was used to treat both cells lines, and the lipogenic genes remained overexpressed in the HB4aC5.2 but not HB4a cells. DHA treatment inhibited all lipogenic genes (except for FABP4) in both cell lines yet only induced death in the HB4aC5.2 cells, mainly when associated with trastuzumab. Neither trastuzumab nor GW9662 alone was able to induce cell death. In conclusion, oncogenic transformation of breast cells by HER2 overexpression may require a reprogramming of lipogenic genetic that is independent of mTORC1 pathway and PPARγ activity. This reprogramming was inhibited by DHA.

Docosahexaenoic Acid Modulates a HER2-Associated Lipogenic Phenotype, Induces Apoptosis, and Increases Trastuzumab Action in HER2-Overexpressing Breast Carcinoma Cells

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Graziela Rosa Ravacci

2015-01-01

Full Text Available In breast cancer, lipid metabolic alterations have been recognized as potential oncogenic stimuli that may promote malignancy. To investigate whether the oncogenic nature of lipogenesis closely depends on the overexpression of HER2 protooncogene, the normal breast cell line, HB4a, was transfected with HER2 cDNA to obtain HER2-overexpressing HB4aC5.2 cells. Both cell lines were treated with trastuzumab and docosahexaenoic acid. HER2 overexpression was accompanied by an increase in the expression of lipogenic genes involved in uptake (CD36, transport (FABP4, and storage (DGAT of exogenous fatty acids (FA, as well as increased activation of “de novo” FA synthesis (FASN. We further investigate whether this lipogenesis reprogramming might be regulated by mTOR/PPARγ pathway. Inhibition of the mTORC1 pathway markers, p70S6 K1, SREBP1, and LIPIN1, as well as an increase in DEPTOR expression (the main inhibitor of the mTOR was detected in HB4aC5.2. Based on these results, a PPARγ selective antagonist, GW9662, was used to treat both cells lines, and the lipogenic genes remained overexpressed in the HB4aC5.2 but not HB4a cells. DHA treatment inhibited all lipogenic genes (except for FABP4 in both cell lines yet only induced death in the HB4aC5.2 cells, mainly when associated with trastuzumab. Neither trastuzumab nor GW9662 alone was able to induce cell death. In conclusion, oncogenic transformation of breast cells by HER2 overexpression may require a reprogramming of lipogenic genetic that is independent of mTORC1 pathway and PPARγ activity. This reprogramming was inhibited by DHA.

L-Proline, GABA Synthesis and Gamma Oscillations in Schizophrenia

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Volk, David W.; Gonzalez-Burgos, Guillermo; Lewis, David A.

2016-01-01

Altered inhibition from parvalbumin-containing GABA neurons is thought to contribute to impaired gamma frequency oscillations and cognitive deficits in schizophrenia. Crabtree and colleagues report that proline dehydrogenase deficits produce excessive cytosolic levels of the GABA-mimetic L-proline which impairs GABA synthesis and gamma oscillations in a manner that mimics schizophrenia.

POD-1/Tcf21 overexpression reduces endogenous SF-1 and StAR expression in rat adrenal cells

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M. M. França

2015-12-01

Full Text Available During gonad and adrenal development, the POD-1/capsulin/TCF21transcription factor negatively regulates SF-1/NR5A1expression, with higher SF-1 levels being associated with increased adrenal cell proliferation and tumorigenesis. In adrenocortical tumor cells, POD-1 binds to the SF-1 E-box promoter region, decreasing SF-1 expression. However, the modulation of SF-1 expression by POD-1 has not previously been described in normal adrenal cells. Here, we analyzed the basal expression of Pod-1 and Sf-1 in primary cultures of glomerulosa (G and fasciculata/reticularis (F/R cells isolated from male Sprague-Dawley rats, and investigated whether POD-1 overexpression modulates the expression of endogenous Sf-1 and its target genes in these cells. POD-1 overexpression, following the transfection of pCMVMycPod-1, significantly decreased the endogenous levels of Sf-1 mRNA and protein in F/R cells, but not in G cells, and also decreased the expression of the SF-1 target StAR in F/R cells. In G cells overexpressing POD-1, no modulation of the expression of SF-1 targets, StAR and CYP11B2, was observed. Our data showing that G and F/R cells respond differently to ectopic POD-1 expression emphasize the functional differences between the outer and inner zones of the adrenal cortex, and support the hypothesis that SF-1 is regulated by POD-1/Tcf21 in normal adrenocortical cells lacking the alterations in cellular physiology found in tumor cells.

Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins

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Cheng Chung-Hsien

2009-07-01

Full Text Available Abstract Background Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase, were overexpressed as double-tagged gene fusions. Both were tagged with glutathione S-transferase (GST at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D, and the other with five contiguous arginine residues (5R. Results Both fusion proteins were overexpressed in Escherichia coli and retained enzymatic activity. The fusions were designed so their surfaces were charged under enzyme reaction conditions, which allowed isolation and immobilization in a single step, through a simple capture with either an anionic or a cationic exchanger (Sepharose Q or Sepharose SP that electrostatically bound the 5D or 5R tag. The introduction of double tags only marginally altered the affinity of the enzymes for their substrates, and the double-tagged proteins were enzymatically active in both soluble and immobilized forms. Combined use of the fusion proteins led to the production of N-acetyl-D-neuraminic acid (Neu5Ac from N-acetyl-D-glucosamine (GlcNAc. Conclusion Double-tagged gene fusions were overexpressed to yield two enzymes that perform sequential steps in sialic acid synthesis. The proteins were easily immobilized via ionic tags onto ionic exchange resins and could thus be purified by direct capture from crude protein extracts. The immobilized, double-tagged proteins were effective for one-pot enzymatic production of sialic acid.

Allergy to fish parvalbumins: studies on the cross-reactivity of allergens from 9 commonly consumed fish.

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Van Do, Thien; Elsayed, Said; Florvaag, Erik; Hordvik, Ivar; Endresen, Curt

2005-12-01

Fish-hypersensitive patients can probably tolerate some fish species while being allergic to others. To determine the allergenic cross-reactivity between 9 commonly edible fish: cod, salmon, pollack, mackerel, tuna, herring, wolffish, halibut, and flounder. Sera from 10 patients allergic to fish and rabbit antisera against 3 parvalbumins (Gad c 1, Sal s 1, and The c 1) were used. Cross-reactivity was investigated by SDS/PAGE and IgE immunoblotting, IgG ELISA, IgE ELISA inhibition, and skin prick test (SPT). Cod (Gad c 1), salmon (Sal s 1), pollack (The c 1), herring, and wolffish share antigenic and allergenic determinants as shown by immunoblots and IgE ELISA, whereas halibut, flounder, tuna, and mackerel displayed lowest cross-reactivities. The highest mean IgE ELISA inhibition percent of 10 sera was obtained by Gad c 1, followed by The c 1, herring, Sal s 1, wolffish, halibut, flounder, tuna, and mackerel with the least inhibition. Nine of the 10 patients showed positive SPT to cod, salmon, and pollack; 8 patients reacted to recombinant (r) Sal s 1. Positive SPTs to rGad c 1 and rThe c 1 were demonstrated in 1 patient. Gad c 1, Sal s 1, The c 1, herring, and wolffish contained the most potent cross-reacting allergens, whereas halibut, flounder, tuna, and mackerel were the least allergenic in the current study. The latter could probably be tolerated by some of the tested patients.

Dual oxidase maturation factor 1 (DUOXA1) overexpression increases reactive oxygen species production and inhibits murine muscle satellite cell differentiation.

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Sandiford, Shelley D E; Kennedy, Karen A M; Xie, Xiaojun; Pickering, J Geoffrey; Li, Shawn S C

2014-01-11

Dual oxidase maturation factor 1 (DUOXA1) has been associated with the maturation of the reactive oxygen species (ROS) producing enzyme, dual oxidase 1 (DUOX1) in the adult thyroid. However, ROS have also been implicated in the development of several tissues. We found that activated muscle satellite cells and primary myoblasts isolated from mice express robust levels of DUOXA1 and that its levels are altered as cells differentiate. To determine whether DUOXA1 levels affect muscle differentiation, we used an adenoviral construct (pCMV5-DUOXA1-GFP) to drive constitutive overexpression of this protein in primary myoblasts. High levels of DUOXA1 throughout myogenesis resulted in enhanced H2O2 production, fusion defects, reduced expression of early (myogenin) and late (myosin heavy chain) markers of differentiation, and elevated levels of apoptosis compared to control cells infected with an empty adenoviral vector (pCMV5-GFP). DUOXA1 knockdown (using a DUOXA1 shRNA construct) resulted in enhanced differentiation compared to cells subjected to a control shRNA, and subjecting DUOXA1 overexpressing cells to siRNAs targeting DUOX1 or apoptosis signal-regulating kinase 1 (ASK1) rescued the phenotype. This study represents the first to demonstrate the importance of DUOXA1 in skeletal muscle myoblasts and that DUOXA1 overexpression in muscle stem cells induces apoptosis and inhibits differentiation through DUOX1 and ASK1.

Mild Oxidative Damage in the Diabetic Rat Heart Is Attenuated by Glyoxalase-1 Overexpression

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Casper G. Schalkwijk

2013-07-01

Full Text Available Diabetes significantly increases the risk of heart failure. The increase in advanced glycation endproducts (AGEs and oxidative stress have been associated with diabetic cardiomyopathy. We recently demonstrated that there is a direct link between AGEs and oxidative stress. Therefore, the aim of the current study was to investigate if a reduction of AGEs by overexpression of the glycation precursor detoxifying enzyme glyoxalase-I (GLO-I can prevent diabetes-induced oxidative damage, inflammation and fibrosis in the heart. Diabetes was induced in wild-type and GLO-I transgenic rats by streptozotocin. After 24-weeks of diabetes, cardiac function was monitored with ultrasound under isoflurane anesthesia. Blood was drawn and heart tissue was collected for further analysis. Analysis with UPLC-MSMS showed that the AGE Nε-(1-carboxymethyllysine and its precursor 3-deoxyglucosone were significantly elevated in the diabetic hearts. Markers of oxidative damage, inflammation, and fibrosis were mildly up-regulated in the heart of the diabetic rats and were attenuated by GLO-I overexpression. In this model of diabetes, these processes were not accompanied by significant changes in systolic heart function, i.e., stroke volume, fractional shortening and ejection fraction. This study shows that 24-weeks of diabetes in rats induce early signs of mild cardiac alterations as indicated by an increase of oxidative stress, inflammation and fibrosis which are mediated, at least partially, by glycation.

Senescence-Induced Alterations of Laminin Chain Expression Modulate Tumorigenicity of Prostate Cancer Cells1

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Sprenger, Cynthia C T; Drivdahl, Rolf H; Woodke, Lillie B; Eyman, Daniel; Reed, May J; Carter, William G; Plymate, Stephen R

2008-01-01

Prostate cancer is an age-associated epithelial cancer, and as such, it contributes significantly to the mortality of the elderly. Senescence is one possible mechanism by which the body defends itself against various epithelial cancers. Senescent cells alter the microenvironment, in part, through changes to the extracellular matrix. Laminins (LMs) are extracellular proteins important to both the structure and function of the microenvironment. Overexpression of the senescence-associated gene mac25 in human prostate cancer cells resulted in increased mRNA levels of the LM α4 and β2 chains compared to empty vector control cells. The purpose of this study was to examine the effects of these senescence-induced LM chains on tumorigenicity of prostate cancer cells. We created stable M12 human prostate cancer lines overexpressing either the LM α4 or β2 chain or both chains. Increased expression of either the LM α4 or β2 chain resulted in increased in vitro migration and in vivo tumorigenicity of those cells, whereas high expression of both chains led to decreased in vitro proliferation and in vivo tumorigenicity compared to M12 control cells. This study demonstrates that senescent prostate epithelial cells can alter the microenvironment and that these changes modulate progression of prostate cancer. PMID:19048114

Methylation alterations are not a major cause of PTTG1 missregulation

International Nuclear Information System (INIS)

Hidalgo, Manuel; Royo, Jose Luis; Galan, Jose Jorge; Sáez, Carmen; Ferrero, Eduardo; Castilla, Carolina; Ramirez-Lorca, Reposo; Pelaez, Pablo; Ruiz, Agustin; Japón, Miguel A

2008-01-01

On its physiological cellular context, PTTG1 controls sister chromatid segregation during mitosis. Within its crosstalk to the cellular arrest machinery, relies a checkpoint of integrity for which gained the over name of securin. PTTG1 was found to promote malignant transformation in 3T3 fibroblasts, and further found to be overexpressed in different tumor types. More recently, PTTG1 has been also related to different processes such as DNA repair and found to trans-activate different cellular pathways involving c-myc, bax or p53, among others. PTTG1 over-expression has been correlated to a worse prognosis in thyroid, lung, colorectal cancer patients, and it can not be excluded that this effect may also occur in other tumor types. Despite the clinical relevance and the increasing molecular characterization of PTTG1, the reason for its up-regulation remains unclear. We analysed PTTG1 differential expression in PC-3, DU-145 and LNCaP tumor cell lines, cultured in the presence of the methyl-transferase inhibitor 5-Aza-2'-deoxycytidine. We also tested whether the CpG island mapping PTTG1 proximal promoter evidenced a differential methylation pattern in differentiated thyroid cancer biopsies concordant to their PTTG1 immunohistochemistry status. Finally, we performed whole-genome LOH studies using Affymetix 50 K microarray technology and FRET analysis to search for allelic imbalances comprising the PTTG1 locus. Our data suggest that neither methylation alterations nor LOH are involved in PTTG1 over-expression. These data, together with those previously reported, point towards a post-transcriptional level of missregulation associated to PTTG1 over-expression

Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA gyrase protection.

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Ruiz, Joaquim

2003-05-01

Quinolones are broad-spectrum antibacterial agents, commonly used in both clinical and veterinary medicine. Their extensive use has resulted in bacteria rapidly developing resistance to these agents. Two mechanisms of quinolone resistance have been established to date: alterations in the targets of quinolones, and decreased accumulation due to impermeability of the membrane and/or an overexpression of efflux pump systems. Recently, mobile elements have also been described, carrying the qnr gene, which confers resistance to quinolones.

Green tea polyphenols rescue of brain defects induced by overexpression of DYRK1A.

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Fayçal Guedj

Full Text Available Individuals with partial HSA21 trisomies and mice with partial MMU16 trisomies containing an extra copy of the DYRK1A gene present various alterations in brain morphogenesis. They present also learning impairments modeling those encountered in Down syndrome. Previous MRI and histological analyses of a transgenic mice generated using a human YAC construct that contains five genes including DYRK1A reveal that DYRK1A is involved, during development, in the control of brain volume and cell density of specific brain regions. Gene dosage correction induces a rescue of the brain volume alterations. DYRK1A is also involved in the control of synaptic plasticity and memory consolidation. Increased gene dosage results in brain morphogenesis defects, low BDNF levels and mnemonic deficits in these mice. Epigallocatechin gallate (EGCG - a member of a natural polyphenols family, found in great amount in green tea leaves - is a specific and safe DYRK1A inhibitor. We maintained control and transgenic mice overexpressing DYRK1A on two different polyphenol-based diets, from gestation to adulthood. The major features of the transgenic phenotype were rescued in these mice.

Dual-Specificity Phosphatase 4 Overexpression in Cells Prevents Hypoxia/Reoxygenation-Induced Apoptosis via the Upregulation of eNOS

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Dougherty, Julie A.; Kilbane Myers, Joanna; Khan, Mahmood; Angelos, Mark G.; Chen, Chun-An

2017-01-01

Mitogen-activated protein kinases (MAPKs) signaling cascades regulate several cellular functions, including differentiation, proliferation, survival, and apoptosis. The duration and magnitude of phosphorylation of these MAPKs are decisive determinants of their physiological functions. Dual-specificity phosphatases exert kinetic control over these signaling cascades. Previously, we demonstrated that DUSP4−/− hearts sustain a larger infarct and have poor functional recovery, when isolated hearts were subjected to ischemia/reperfusion. Uncontrolled p38 activation and upregulation of Nox4 expression are the main effectors for this functional alteration. Here, dual-specificity phosphatase 4 (DUSP4) overexpression in endothelial cells was used to investigate the role of DUSP4 on the modulation of reactive oxygen species (ROS) generation and vascular function, when cells were subjected to hypoxia/reoxygenation (H/R) insult. Immunostaining with cleaved caspase-3 revealed that DUSP4 overexpression prevents caspase-3 activation and apoptosis after H/R. The beneficial effects occur via modulating p38 activity, increased NO bioavailability, and reduced oxidative stress. More importantly, DUSP4 overexpression upregulates eNOS protein expression (1.62 ± 0.33 versus 0.65 ± 0.16) during H/R-induced stress. NO is a critical small molecule involved in regulating vascular tone, vascular growth, platelet aggregation, and modulation of inflammation. The level of NO generation determined using DAF-2 fluorescence demonstrated that DUSP4 overexpression augments NO production and thus improves vascular function. The level of superoxide generated from cells after being subjected to H/R was determined using dihydroethidium-HPLC method. The results suggested that DUSP4 overexpression in cells decreases H/R-induced superoxide generation (1.56 ± 0.14 versus 1.19 ± 0.05) and thus reduces oxidant stress. This also correlates with the reduction in the total protein S

The effect of aquaporin 5 overexpression on the Ras signaling pathway

International Nuclear Information System (INIS)

Woo, Janghee; Lee, Juna; Kim, Myoung Sook; Jang, Se Jin; Sidransky, David; Moon, Chulso

2008-01-01

Human aquaporin 5 (AQP5) has been shown to be overexpressed in multiple cancers, such as pancreatic cancer and colon cancer. Furthermore, it has been reported that ectopic expression of AQP5 leads to many phenotypic changes characteristic of transformation. However, the biochemical mechanism leading to transformation in AQP5-overexpressing cells has not been clearly elucidated. In this report, the overexpression of AQP5 in NIH3T3 cells demonstrated a significant effect on Ras activity and, thus, cell proliferation. Furthermore, this influence was shown to be mediated by phosphorylation of the PKA consensus site of AQP5. This is the first evidence demonstrating an association between AQP5 and a signaling pathway, namely the Ras signal transduction pathway, which may be the basis of the oncogenic properties seen in AQP-overexpressing cells

Overexpression of the steroidogenic enzyme cytochrome P450 side chain cleavage in the ventral tegmental area increases 3α,5α-THP and reduces long-term operant ethanol self-administration.

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Cook, Jason B; Werner, David F; Maldonado-Devincci, Antoniette M; Leonard, Maggie N; Fisher, Kristen R; O'Buckley, Todd K; Porcu, Patrizia; McCown, Thomas J; Besheer, Joyce; Hodge, Clyde W; Morrow, A Leslie

2014-04-23

Neuroactive steroids are endogenous neuromodulators capable of altering neuronal activity and behavior. In rodents, systemic administration of endogenous or synthetic neuroactive steroids reduces ethanol self-administration. We hypothesized this effect arises from actions within mesolimbic brain regions that we targeted by viral gene delivery. Cytochrome P450 side chain cleavage (P450scc) converts cholesterol to pregnenolone, the rate-limiting enzymatic reaction in neurosteroidogenesis. Therefore, we constructed a recombinant adeno-associated serotype 2 viral vector (rAAV2), which drives P450scc expression and neuroactive steroid synthesis. The P450scc-expressing vector (rAAV2-P450scc) or control GFP-expressing vector (rAAV2-GFP) were injected bilaterally into the ventral tegmental area (VTA) or nucleus accumbens (NAc) of alcohol preferring (P) rats trained to self-administer ethanol. P450scc overexpression in the VTA significantly reduced ethanol self-administration by 20% over the 3 week test period. P450scc overexpression in the NAc, however, did not alter ethanol self-administration. Locomotor activity was unaltered by vector administration to either region. P450scc overexpression produced a 36% increase in (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone)-positive cells in the VTA, but did not increase 3α,5α-THP immunoreactivity in NAc. These results suggest that P450scc overexpression and the resultant increase of 3α,5α-THP-positive cells in the VTA reduces ethanol reinforcement. 3α,5α-THP is localized to neurons in the VTA, including tyrosine hydroxylase neurons, but not astrocytes. Overall, the results demonstrate that using gene delivery to modulate neuroactive steroids shows promise for examining the neuronal mechanisms of moderate ethanol drinking, which could be extended to other behavioral paradigms and neuropsychiatric pathology.

DNA fingerprinting tags novel altered chromosomal regions and identifies the involvement of SOX5 in the progression of prostate cancer.

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Ma, Stephanie; Chan, Yuen Piu; Woolcock, Bruce; Hu, Liang; Wong, Kai Yau; Ling, Ming Tat; Bainbridge, Terry; Webber, Douglas; Chan, Tim Hon Man; Guan, Xin-Yuan; Lam, Wan; Vielkind, Juergen; Chan, Kwok Wah

2009-05-15

Identification of genomic alterations associated with the progression of prostate cancer may facilitate the better understanding of the development of this highly variable disease. Matched normal, premalignant high-grade prostatic intraepithelial neoplasia and invasive prostate carcinoma cells were procured by laser capture microdissection (LCM) from human radical prostatectomy specimens. From these cells, comparative DNA fingerprints were generated by a modified PCR-based technique called scanning of microdissected archival lesion (SMAL)-PCR. Recurrent polymorphic fingerprint fragments were used in tagging altered chromosomal regions. Altered regions were found at cytobands 1p31.3, 1q44, 2p23.1, 3p26.3, 3q22.3, 4q22.3, 4q35.2, 5q23.2, 8q22.3, 8q24.13, 9q21.3, 9q22.32, 10q11.21, 11p13, 12p12.1, 13q12.1, 16q12.2 and 18q21.31. Candidate genes in the surrounding area that may possibly harbor mutations that change normal prostatic cells to progress into their tumor stages were proposed. Of these fragments, a 420 bp alteration, absent in all 26 normal samples screened, was observed in 2 tumors. This fragment was cloned, sequenced and localized to chromosome 12p12.1. Within this region, candidate gene sex determining region Y-box 5 (SOX5) was proposed. Further studies of SOX5 in cell lines, xenografts and human prostate specimens, at both the RNA and protein levels, found overexpression of the gene in tumors. This overexpression was then subsequently found by fluorescent in situ hybridization to be caused by amplification of the region. In conclusion, our results suggest LCM coupled with SMAL-PCR DNA fingerprinting is a useful method for the screening and identification of chromosomal regions and genes associated with cancer development. Further, overexpression of SOX5 is associated with prostate tumor progression and early development of distant metastasis. (c) 2008 Wiley-Liss, Inc.

ERBB-2 overexpression as a risk factor for malignant phaeochromocytomas and paraganglinomas.

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Wang, Weiqing; Zhong, Xu; Ye, Lei; Qi, Yan; Su, TingWei; Wei, Qing; Xie, Jing; Jiang, Lei; Jiang, Yiran; Zhou, Weiwei; Cui, Bin; Ning, Guang

2016-06-01

There are currently no good histological or molecular markers to differentiate benign from malignant phaeochromocytomas and paraganglinomas (PPGLs). Our previous cross-sectional study observed that ERBB-2 overexpression was associated with malignant PPGLs. This study aimed to evaluate the predictive value of ERBB-2 overexpression for metastasis in PPGLs in a large population. A total of 262 patients diagnosed as PPGLs in our institution between 2002 and 2012 were included. We analysed ERBB-2 protein expression in the primary PPGL tumours by immunohistochemistry (IHC) and ERBB-2 amplification by fluorescence in situ hybridization (FISH). Direct Sanger sequencing was performed to examine ERBB-2 exon 20 mutations. The occurrence of malignant PPGLs was documented in the follow-up period. Kaplan-Meier analysis and Cox proportional hazard models were used to evaluate the association between ERBB-2 overexpression and metastasis of PPGLs. Twenty-six (9·9%) patients had ERBB-2 overexpression in their primary PPGL tumours, which was significantly associated with ERBB-2 amplification (17/25, 68%). No ERBB-2 mutation was found. At a median follow-up of 4·5 years, a total of 23 malignant PPGLs were documented, including eight (30·8%) patients in the ERBB-2 overexpression group and 15 (6·4%) patients in the ERBB-2-negative group. The incidence rate of metastasis was 5·3 per 100 person-years vs 1·4 per 100 person-years in the ERBB-2 overexpression and ERBB-2-negative groups (P overexpression was associated with decreased metastasis-free survival (P = 0·001, log-rank test). After adjusting for primary tumour size and location, Cox regression analysis revealed that ERBB-2 overexpression was independently associated with risk of malignant PPGLs (HR = 2·78; 95% CI, 1·12-6·90; P = 0·028). Patients harbouring tumours with ERBB-2 overexpression have a significantly higher risk of developing malignant PPGLs. © 2016 John Wiley & Sons Ltd.

Frequent alteration of MDM2 and p53 in the molecular progression of recurring non-Hodgkin's lymphoma

DEFF Research Database (Denmark)

Møller, Michael Boe; Nielsen, O; Pedersen, Niels Tinggaard

2002-01-01

-Hodgkin's lymphoma. METHODS AND RESULTS: We have analysed sequential biopsies from 42 non-Hodgkin's lymphoma patients immunohistochemically for p53 alterations (based on p53 and p21Waf1 expression), as well as for expression of MDM2, p27Kip1 and cyclin D3. Relapse of follicle centre lymphoma was associated with p53...... alterations as 5/6 (83%) follicle centre lymphomas with normal p53 at diagnosis showed p53 alterations at relapse. Of these cases, three showed transformation to diffuse large B-cell lymphoma. p53 alteration was also associated with relapse of de novo diffuse large B-cell lymphoma and T-cell non......-Hodgkin's lymphoma, as 2/5 (40%) diffuse large B-cell lymphomas and 3/9 (33%) T-cell non-Hodgkin's lymphomas with normal p53 at diagnosis showed p53 alterations at relapse. No indolent non-Hodgkin's lymphoma case showed MDM2 over-expression at diagnosis, whereas 4/5 (80%) transformed diffuse large B-cell lymphomas...

Poly (A+ transcriptome assessment of ERBB2-induced alterations in breast cell lines.

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Dirce Maria Carraro

Full Text Available We report the first quantitative and qualitative analysis of the poly (A⁺ transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in ERBB2 over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as LOX, ATP5L, GALNT3, and MME revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different ERBB2 backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by ERBB2 overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.

Overexpression of pucC improves the heterologous protein expression level in a Rhodobacter sphaeroides expression system.

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Cheng, L; Chen, G; Ding, G; Zhao, Z; Dong, T; Hu, Z

2015-04-27

The Rhodobacter sphaeroides system has been used to express membrane proteins. However, its low yield has substantially limited its application. In order to promote the protein expression capability of this system, the pucC gene, which plays a crucial role in assembling the R. sphaeroides light-harvesting 2 complex (LH2), was overexpressed. To build a pucC overexpression strain, a pucC overexpression vector was constructed and transformed into R. sphaeroides CQU68. The overexpression efficiency was evaluated by quantitative real-time polymerase chain reaction. A well-used reporter β-glucuronidase (GUS) was fusion-expressed with LH2 to evaluate the heterologous protein expression level. As a result, the cell culture and protein in the pucC overexpression strain showed much higher typical spectral absorption peaks at 800 and 850 nm compared with the non-overexpression strain, suggesting a higher expression level of LH2-GUS fusion protein in the pucC overexpression strain. This result was further confirmed by Western blot, which also showed a much higher level of heterologous protein expression in the pucC overexpression strain. We further compared GUS activity in pucC overexpression and non-overexpression strains, the results of which showed that GUS activity in the pucC overexpression strain was approximately ten-fold that in the non-overexpression strain. These results demonstrate that overexpressed pucC can promote heterologous protein expression levels in R. sphaeroides.

Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism

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Karhumaa Kaisa

2011-07-01

Full Text Available Abstract Background Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric n-butanol. Saccharomyces cerevisiae was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials. Results The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in anaerobic fermentation of glucose in mineral medium in S. cerevisiae. Isobutanol yield was further improved by twofold by the additional overexpression of BAT2, encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of ILV6, encoding the regulatory subunit of Ilv2, in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the ILV2 ILV3 ILV5 overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD complex medium under aerobic conditions, respectively. Conclusions Overexpression of genes ILV2, ILV3, ILV5, and BAT2 in valine metabolism led to an increase in isobutanol production in S. cerevisiae. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of ILV2, ILV3, and ILV5 on isobutanol production. Aerobic cultivations of the ILV2 ILV3 ILV5 overexpression strain and the reference strain showed that supplying amino acids in cultivation media

PRDM14 is expressed in germ cell tumors with constitutive overexpression altering human germline differentiation and proliferation

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Joanna J. Gell

2018-03-01

Full Text Available Germ cell tumors (GCTs are a heterogeneous group of tumors occurring in gonadal and extragonadal locations. GCTs are hypothesized to arise from primordial germ cells (PGCs, which fail to differentiate. One recently identified susceptibility loci for human GCT is PR (PRDI-BF1 and RIZ domain proteins 14 (PRDM14. PRDM14 is expressed in early primate PGCs and is repressed as PGCs differentiate. To examine PRDM14 in human GCTs we profiled human GCT cell lines and patient samples and discovered that PRDM14 is expressed in embryonal carcinoma cell lines, embryonal carcinomas, seminomas, intracranial germinomas and yolk sac tumors, but is not expressed in teratomas. To model constitutive overexpression in human PGCs, we generated PGC-like cells (PGCLCs from human pluripotent stem cells (PSCs and discovered that elevated expression of PRDM14 does not block early PGC formation. Instead, we show that elevated PRDM14 in PGCLCs causes proliferation and differentiation defects in the germline. Keywords: Germ cell tumor, PRDM14, Cell differentiation, Primordial germ cell, Proliferation

Effect of SOCS1 overexpression on RPE cell activation by proinflammatory cytokines.

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Bazewicz, Magdalena; Draganova, Dafina; Makhoul, Maya; Chtarto, Abdel; Elmaleh, Valerie; Tenenbaum, Liliane; Caspers, Laure; Bruyns, Catherine; Willermain, François

2016-09-06

The purpose of this study was to investigate the in vitro effect of Suppressor Of Cytokine Signaling 1 (SOCS1) overexpression in retinal pigment epithelium (RPE) cells on their activation by pro-inflammatory cytokines IFNγ, TNFα and IL-17. Retinal pigment epithelium cells (ARPE-19) were stably transfected with the control plasmid pIRES2-AcGFP1 or the plasmid pSOCS1-IRES2-AcGFP1. They were stimulated by IFNγ (150ng/ml), TNFα (30ng/ml) or IL-17 (100ng/ml). The levels of SOCS1 mRNA were measured by real-time PCR. Signal Transducer and Activator of Transcription 1 (STAT1) phosphorylation and IκBα expression were analysed by western Blot (WB). IL-8 secretion was analysed by ELISA and expression of MHCII molecules and ICAM-1/CD54 by flow cytometry. Our data show that SOCS1 mRNA overexpression in RPE cells prevents IFNγ-induced SOCS1 mRNA increase and IFNγ-mediated STAT1 phosphorylation. Moreover, SOCS1 overexpression in RPE cells inhibits IFNγ-induced decrease of IL-8 secretion and prevents IFNγ-induced MHC II and ICAM1/CD54 upregulation. However, SOCS1 overexpression does not affect TNFα-induced IκBα degradation nor block TNFα-induced or IL-17-induced IL-8 secretion. On the contrary, IL-17-induced secretion is increased by SOCS1 overexpression. In conclusion, SOCS1 overexpression in RPE cells inhibits some IFNγ-mediated responses that lead to uveitis development. This notion raises the possibility that SOCS1 overexpression could be a novel target for treating non-infectious uveitis. However, some proinflammatory effects of TNFα and IL-17 stimulation on RPE are not blocked by SOCS1 overexpression. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

DEK protein overexpression predicts poor prognosis in pancreatic ductal adenocarcinoma.

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Sun, Jie; Bi, Fangfang; Yang, Yang; Zhang, Yuan; Jin, Aihua; Li, Jinzi; Lin, Zhenhua

2017-02-01

DEK, a transcription factor, is involved in mRNA splicing, transcriptional control, cell division and differentiation. Recent studies suggest that DEK overexpression can promote tumorigenesis in a wide range of cancer cell types. However, little is known concerning the status of DEK in pancreatic ductal adenocarcinoma (PDAC). Based on the microarray data from Gene Expression Omnibus (GEO), the expression levels of DEK mRNA in PDAC tissues were significantly higher than levels in the adjacent non-tumor tissues. To explore the clinical features of DEK overexpression in PDAC, 87 PDAC and 52 normal pancreas tissues were selected for immunoenzyme staining of the DEK protein. Localization of the DEK protein was detected in PANC-1 pancreatic cancer cells using immunofluorescence (IF) staining. The correlations between DEK overexpression and the clinical features of PDAC were evaluated using the Chi-squared (χ2) and Fisher's exact tests. The survival rates were calculated by the Kaplan-Meier method, and the relationship between prognostic factors and patient survival was also analyzed by the Cox proportional hazard models. The expression levels of DEK mRNA in PDAC tissues were significantly higher than that in the adjacent non‑tumor tissues. The DEK protein showed a primarily nuclear staining pattern in PDAC. The positive rate of the DEK protein was 52.9% (46/87) in PDAC, which was significantly higher than that in the adjacent normal pancreatic tissues (7.7%, 4/52). DEK overexpression in PDAC was correlated with tumor size, histological grade, tumor‑node‑metastasis (TNM) stage and overall survival (OS) rates. In addition, multivariate analysis demonstrated that DEK overexpression was an independent prognostic factor along with histological grade and TNM stage in patients with PDAC. In conclusion, DEK overexpression is associated with PDAC progression and may be a potential biomarker for poor prognostic evaluation in PDAC.

CD147 overexpression promotes tumorigenicity in Chinese hamster ovary cells.

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Yong, Yu-Le; Liao, Cheng-Gong; Wei, Ding; Chen, Zhi-Nan; Bian, Huijie

2016-04-01

CD147 overexpresses in many epithelium-originated tumors and plays an important role in tumor migration and invasion. Most studies aim at the role of CD147 in tumor progression using tumor cell models. However, the influence of abnormal overexpression of CD147 on neoplastic transformation of normal cells is unknown. Here, the role of CD147 in malignant phenotype transformation in CHO cells was investigated. Three CHO cell lines that stably overexpressed CD147 (CHO-CD147), EGFP-CD147 (CHO-EGFP-CD147), and EGFP (CHO-EGFP) were generated by transfection of plasmids containing human CD147, EGFP-human CD147, and EGFP genes into CHO cells. Cell migration and invasion were detected by wound healing and transwell matrix penetration assay. Trypan blue exclusion, MTT, cell cycle analysis, and BrdU cell proliferation assay were used to detect cell viability and cell proliferation. Annexin V-FITC analysis was performed to detect apoptosis. We found that CD147 overexpression promoted the migration and invasion of CHO cells. CD147 accelerated the G1 to S phase transition and enhanced the CHO cell proliferation. Overexpression of CD147 inhibited both early- and late-stages of apoptosis of CHO-CD147 cells, which is caused by serum deprivation. CHO-EGFP-CD147 cells showed an increased anchorage-independent growth compared with CHO-EGFP cells as detected by soft-agar colony formation assay. The tumors formed by CHO-CD147 cells in nude mice were larger and coupled with higher expression of proliferating cell nuclear antigen and Ki-67 than that of CHO cells. In conclusion, human CD147 overexpression induces malignant phenotype in CHO cells. © 2015 International Federation for Cell Biology.

Brain phenotype of transgenic mice overexpressing cystathionine β-synthase.

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Vinciane Régnier

Full Text Available The cystathionine β-synthase (CBS gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA metabolism, a pathway important for several brain physiological processes.Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1 expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line.We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.

Brain phenotype of transgenic mice overexpressing cystathionine β-synthase.

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Régnier, Vinciane; Billard, Jean-Marie; Gupta, Sapna; Potier, Brigitte; Woerner, Stéphanie; Paly, Evelyne; Ledru, Aurélie; David, Sabrina; Luilier, Sabrina; Bizot, Jean-Charles; Vacano, Guido; Kraus, Jan P; Patterson, David; Kruger, Warren D; Delabar, Jean M; London, Jaqueline

2012-01-01

The cystathionine β-synthase (CBS) gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS) cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA) metabolism, a pathway important for several brain physiological processes. Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1) expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line. We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.

WDR62 overexpression is associated with a poor prognosis in patients with lung adenocarcinoma.

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Shinmura, Kazuya; Kato, Hisami; Kawanishi, Yuichi; Igarashi, Hisaki; Inoue, Yusuke; Yoshimura, Katsuhiro; Nakamura, Satoki; Fujita, Hidehiko; Funai, Kazuhito; Tanahashi, Masayuki; Niwa, Hiroshi; Ogawa, Hiroshi; Sugimura, Haruhiko

2017-08-01

Human WDR62, which is localized in the cytoplasm including the centrosome, is known to be responsible for primary microcephaly; however, the role of WDR62 abnormality in cancers remains largely unknown. In this study, we aimed to reveal the pathological role of WDR62 abnormality in lung adenocarcinoma (LAC). We first examined the WDR62 mRNA expression level of LAC (n = 64) using a QRT-PCR analysis and found that WDR62 mRNA transcripts were significantly overexpressed in LAC (P = 0.0432, Wilcoxon matched pairs test). An immunohistochemical analysis for LAC (n = 237) showed that WDR62 proteins were also significantly overexpressed in LAC (P overexpression have a short overall survival (P = 0.0378, log-rank test), and a multivariate analysis revealed that WDR62 overexpression was an independent predictor of a poor survival outcome among LAC patients (hazard ratio, 2.032; 95% confidence interval, 1.071-3.777; P = 0.0305). Next, we examined the functional effect of WDR62 overexpression on the lung cancer cell line H1299. WDR62-overexpressing lung cancer cells exhibited an increase in cell growth. Moreover, the concurrent overexpression of WDR62 and TPX2, a WDR62-interacting protein that is also overexpressed in LAC, induced centrosome amplification in the lung cells. Finally, we disclosed that the concurrent overexpression of WDR62 and TPX2 is common in diverse human cancers, using data from the Cancer Genome Atlas. These results suggested that WDR62 overexpression is associated with a poor prognosis in patients with LAC and leads to an increase in the malignant potential of lung cells. © 2017 Wiley Periodicals, Inc.

Overexpression of Myo1e in mouse podocytes enhances cellular endocytosis, migration, and adhesion.

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Jin, Xia; Wang, Wenjing; Mao, Jianhua; Shen, Huijun; Fu, Haidong; Wang, Xia; Gu, Weizhong; Liu, Aimin; Yu, Huimin; Shu, Qiang; Du, Lizhong

2014-02-01

Podocytes are a terminally differentiated and highly specialized cell type in the glomerulus that forms a crucial component of the glomerular filtration barrier. Recently, Myo1e was identified in the podocytes of glomeruli. Myo1e podocyte-specific knockout mice exhibit proteinuria, podocyte foot process effacement, glomerular basement membrane disorganization, signs of chronic renal injury, and kidney inflammation. After overexpression of Myo1e in a conditionally immortalized mouse podocyte cell line (MPC5), podocyte migration was evaluated via transwell assay, endocytosis was evaluated using FITC-transferrin, and adhesion was evaluated using a detachment assay after puromycin aminonucleoside treatment. Myo1e overexpression significantly increased the adherence of podocytes. ANOVA analysis indicated significant differences for cell adhesion between the overexpression and control groups (overexpression vs. control, t = 11.3199, P = 0.005; overexpression vs. negative control, t = 12.0570, P = 0.0006). Overexpression of Myo1e inhibited puromycin aminonucleoside-induced podocyte detachment, and the number of cells remaining on the bottom of the culture plate increased. Cell migration was enhanced in Myo1e-overexpressing podocytes in the transwell migration assay. Internalization of FITC-transferrin also increased in Myo1e-overexpressing podocytes relative to control cells. Overexpression of Myo1e can enhance podocyte migration ability, endocytosis, and attachment to the glomerular basement membrane. Restoration of Myo1e expression in podocytes may therefore strengthen their functional integrity against environmental and mechanical injury. © 2013 Wiley Periodicals, Inc.

Energetic heavy ions overcome tumor radioresistance caused by overexpression of Bcl-2

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Hamada, Nobuyuki; Hara, Takamitsu; Omura-Minamisawa, Motoko; Funayama, Tomoo; Sakashita, Tetsuya; Sora, Sakura; Yokota, Yuichiro; Nakano, Takashi

2008-01-01

Background and purpose: Overexpression of Bcl-2 is frequent in human cancers and has been associated with radioresistance. Here we investigated the potential impact of heavy ions on Bcl-2 overexpressing tumors. Materials and methods: Bcl-2 cells (Bcl-2 overexpressing HeLa cells) and Neo cells (neomycin resistant gene-expressing HeLa cells) exposed to γ-rays or heavy ions were assessed for the clonogenic survival, apoptosis and cell cycle distribution. Results: Whereas Bcl-2 cells were more resistant to γ-rays (0.2 keV/μm) and helium ions (16.2 keV/μm) than Neo cells, heavy ions (76.3-1610 keV/μm) yielded similar survival regardless of Bcl-2 overexpression. Carbon ions (108 keV/μm) decreased the difference in the apoptotic incidence between Bcl-2 and Neo cells, and prolonged G 2 /M arrest that occurred more extensively in Bcl-2 cells than in Neo cells. Conclusions: High-LET heavy ions overcome tumor radioresistance caused by Bcl-2 overexpression, which may be explained at least in part by the enhanced apoptotic response and prolonged G 2 /M arrest. Thus, heavy-ion therapy may be a promising modality for Bcl-2 overexpressing radioresistant tumors

Resveratrol Prevents Cellular and Behavioral Sensory Alterations in the Animal Model of Autism Induced by Valproic Acid

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Mellanie Fontes-Dutra

2018-05-01

Full Text Available Autism spectrum disorder (ASD is characterized by impairments in both social communication and interaction and repetitive or stereotyped behaviors. Although its etiology remains unknown, genetic and environmental risk factors have been associated with this disorder, including the exposure to valproic acid (VPA during pregnancy. Resveratrol (RSV is an anti-inflammatory and antioxidant molecule known to prevent social impairments in the VPA animal model of autism. This study aimed to analyze the effects of prenatal exposure to VPA, as well as possible preventive effects of RSV, on sensory behavior, the localization of GABAergic parvalbumin (PV+ neurons in sensory brain regions and the expression of proteins of excitatory and inhibitory synapses. Pregnant rats were treated daily with RSV (3.6 mg/kg from E6.5 to E18.5 and injected with VPA (600 mg/kg in the E12.5. Male pups were analyzed in Nest Seeking (NS behavior and in whisker nuisance task (WNT. At P30, the tissues were removed and analyzed by immunofluorescence and western blotting. Our data showed for the first time an altered localization of PV+-neurons in primary sensory cortex and amygdala. We also showed a reduced level of gephyrin in the primary somatosensory area (PSSA of VPA animals. The treatment with RSV prevented all the aforementioned alterations triggered by VPA. Our data shed light on the relevance of sensory component in ASD and highlights the interplay between RSV and VPA animal model as an important tool to investigate the pathophysiology of ASD.

Overexpression of FurA in Anabaena sp. PCC 7120 reveals new targets for this regulator involved in photosynthesis, iron uptake and cellular morphology.

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González, Andrés; Bes, M Teresa; Barja, François; Peleato, M Luisa; Fillat, María F

2010-11-01

Previous genomic analyses of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 have identified three ferric uptake regulator (Fur) homologs with low sequence identities and probably different functions in the cell. FurA is a constitutive protein that shares the highest homology with Fur from heterotrophic bacteria and appears to be essential for in vitro growth. In this study, we have analysed the effects of FurA overexpression on the Anabaena sp. phenotype and investigated which of the observed alterations were directly operated by FurA. Overexpression of the regulator led to changes in cellular morphology, resulting in shorter filaments with rounded cells of different sizes. The furA-overexpressing strain showed a slower photoautotrophic growth and a marked decrease in the oxygen evolution rate. Overexpression of the regulator also decreased both catalase and superoxide dismutase activities, but did not lead to an increase in the levels of intracellular reactive oxygen species. By combining phenotypic studies, reverse transcription-PCR analyses and electrophoretic mobility shift assays, we identified three novel direct targets of FurA, including genes encoding a siderophore outer membrane transporter (schT), bacterial actins (mreBCD) and the PSII reaction center protein D1 (psbA). The affinity of FurA for these novel targets was markedly affected by the absence of divalent metal ions, confirming previous evidence of a critical role for the metal co-repressor in the function of the regulator in vivo. The results unravel new cellular processes modulated by FurA, supporting its role as a global transcriptional regulator in Anabaena sp. PCC 7120.

DREADD in parvalbumin interneurons of the dentate gyrus modulates anxiety, social interaction and memory extinction.

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Zou, D; Chen, L; Deng, D; Jiang, D; Dong, F; McSweeney, C; Zhou, Y; Liu, L; Chen, G; Wu, Y; Mao, Y

2016-01-01

Parvalbumin (PV)-positive interneurons in the hippocampus play a critical role in animal memory, such as spatial working memory. However, how PV-positive interneurons in the subregions of the hippocampus affect animal behaviors remains poorly defined. Here, we achieved specific and reversible activation of PV-positive interneurons using designer receptors exclusively activated by designer drugs (DREADD) technology. Inducible DREADD expression was demonstrated in vitro in cultured neurons, in which co-transfection of the hM3D-Gq-mCherry vector with a Cre plasmid resulted in a cellular response to hM3Dq ligand clozapine-N-oxide (CNO) stimulation. In addition, the dentate gyrus (DG) of PV-Cre mice received bilateral injection of control lentivirus or lentivirus expressing double floxed hM3D-Gq-mCherry. Selective activation of PV-positive interneurons in the DG did not affect locomotor activity or depression-related behavior in mice. Interestingly, stimulation of PV-positive interneurons induced an anxiolytic effect. Activation of PVpositive interneurons appears to impair social interaction to novelty, but has no effect on social motivation. However, this defect is likely due to the anxiolytic effect as the exploratory behavior of mice expressing hM3DGq is significantly increased. Mice expressing hM3D-Gq did not affect novel object recognition. Activation of PV-positive interneurons in the DG maintains intact cued and contextual fear memory but facilitates fear extinction. Collectively, our results demonstrated that proper control of PV interneurons activity in the DG is critical for regulation of the anxiety, social interaction and fear extinction. These results improve our fundamental understanding of the physiological role of PV-positive interneurons in the hippocampus.

Overexpression of microRNA-1288 in oesophageal squamous cell carcinoma

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Gopalan, Vinod; Islam, Farhadul; Pillai, Suja; Tang, Johnny Cheuk-On; Tong, Daniel King-Hung; Law, Simon; Chan, Kwok-Wah; Lam, Alfred King-Yin

2016-01-01

Purpose: This study aims to examine the expression profiles miR-1288 in oesophageal squamous cell carcinoma (ESCC). The cellular implications and target interactions of ESCC cells following miR-1288 overexpression was also examined. Methods: In total, 120 oesophageal tissues (90 primary ESCCs and 30 non-neoplastic tissues) were recruited for miR-1288 expression analysis using qRT-PCR. An exogenous miR-1288 mimic and its inhibitor were used to explore the in-vitro effects of miR-1288 on ESCC cells by performing cell proliferation, colony formation, cell invasion and migration assays. Localisation and modulatory changes of various miR-1288 regulated proteins such as FOXO1, p53, TAB3, BCL2 and kRAS was examined using immunofluorescence and western blot. Results: Overexpression of miR-1288 was more often noted in ESCC tissues when compared to non-neoplastic oesophageal tissues. High expression was often noted in high grade carcinomas and with metastases. Patients with high levels of miR-1288 expression showed a slightly better survival compared to patients with low miR-1288 levels. Furthermore, overexpression of miR-1288 showed increased cell proliferation and colony formation, improved cell migration and enhanced cell invasion properties in ESCC cells. In addition, miR-1288 overexpression in ESCC cells showed repression of cytoplasmic tumour suppressor FOXO1 protein expression. Inversely, inhibition of miR-1288 expression exhibited remarkable upregulation of FOXO1 protein, while expressions of other tested proteins remain unchanged. Conclusions: Up regulation of miR-1288 expression in ESCC tissues and miR-1288 induced oncogenic features of ESCC cells in-vitro indicates the oncogenic roles of miR-1288 in ESCCs. Overexpression of miR-1288 play a key role in the pathogenesis of ESCCs and its modulation may have potential therapeutic value in patients with ESCC. - Highlights: • miR-1288 was more often noted in neoplastic than non-neoplastic tissue. • miR-1288

Overexpression of microRNA-1288 in oesophageal squamous cell carcinoma

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Gopalan, Vinod; Islam, Farhadul; Pillai, Suja [Cancer Molecular Pathology, School of Medicine and Menzies Health Institute Queensland, Griffith University, Gold Coast (Australia); Tang, Johnny Cheuk-On [State Key Laboratory of Chirosciences, Lo Ka Chung Centre for Natural Anti-cancer Drug Development, Department of Applied Biology and Chemical Technology, the Hong Kong Polytechnic University (Hong Kong); Tong, Daniel King-Hung; Law, Simon [Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital (Hong Kong); Chan, Kwok-Wah [Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital (Hong Kong); Lam, Alfred King-Yin, E-mail: a.lam@griffith.edu.au [Cancer Molecular Pathology, School of Medicine and Menzies Health Institute Queensland, Griffith University, Gold Coast (Australia)

2016-11-01

Purpose: This study aims to examine the expression profiles miR-1288 in oesophageal squamous cell carcinoma (ESCC). The cellular implications and target interactions of ESCC cells following miR-1288 overexpression was also examined. Methods: In total, 120 oesophageal tissues (90 primary ESCCs and 30 non-neoplastic tissues) were recruited for miR-1288 expression analysis using qRT-PCR. An exogenous miR-1288 mimic and its inhibitor were used to explore the in-vitro effects of miR-1288 on ESCC cells by performing cell proliferation, colony formation, cell invasion and migration assays. Localisation and modulatory changes of various miR-1288 regulated proteins such as FOXO1, p53, TAB3, BCL2 and kRAS was examined using immunofluorescence and western blot. Results: Overexpression of miR-1288 was more often noted in ESCC tissues when compared to non-neoplastic oesophageal tissues. High expression was often noted in high grade carcinomas and with metastases. Patients with high levels of miR-1288 expression showed a slightly better survival compared to patients with low miR-1288 levels. Furthermore, overexpression of miR-1288 showed increased cell proliferation and colony formation, improved cell migration and enhanced cell invasion properties in ESCC cells. In addition, miR-1288 overexpression in ESCC cells showed repression of cytoplasmic tumour suppressor FOXO1 protein expression. Inversely, inhibition of miR-1288 expression exhibited remarkable upregulation of FOXO1 protein, while expressions of other tested proteins remain unchanged. Conclusions: Up regulation of miR-1288 expression in ESCC tissues and miR-1288 induced oncogenic features of ESCC cells in-vitro indicates the oncogenic roles of miR-1288 in ESCCs. Overexpression of miR-1288 play a key role in the pathogenesis of ESCCs and its modulation may have potential therapeutic value in patients with ESCC. - Highlights: • miR-1288 was more often noted in neoplastic than non-neoplastic tissue. • miR-1288

[Effect of luxS overexpression on biofilm formation by Streptococcus mutans].

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He, Zhiyan; Wang, Yuxia; Huang, Zhengwei

2015-09-01

To evaluate the effect of quorum sensing luxS gene on biofilm formation through construction of a luxS overexpression strain by Streptococcus mutans (Sm). In order to construct pIB-luxS plasmid, the luxS gene fragment amplified by PCR was inserted into the shuttle plasmid pIB169 by corresponding double digests. The pIB-luxS plasmid was linearized electro-transformed into Sm cell and the overexpression strain was selected on chloramphenicol plate and testified by electrophoresis and western blot. The growth rate of both Sm wild type strain and its luxS overexpression strain were observed. Methyl thiazolyl tetrazolium (MTT) assay method was used to compare the biofilm formation quantification by both strains at different time points and containing different sucrose. The structures of the biofilms were observed by using confocal laser scanning microscopy, and biofilm-related gene expressions were investigated by real-time PCR. All experiments were performed in triplicate. The luxS overexpression strain was successfully constructed and confirmed by electrophoresis and Western blotting. The planktonic growth mode of the wild-type and luxS overexpression strain showed no difference, but biofilm formed by Sm overexpression strain was 0.400 ± 0.009 and 0.609 ± 0.041 at 14 and 24 h, higher than the wild type strain biofilm at the same time point (0.352 ± 0.028 and 0.533 ± 0.014, respectively, P overexpression strain raised to 1.041 ± 0.038, higher than that by the wild type strain (0.831 ± 0.020, P overexpression strain aggregated into distinct clusters on structure, genes expression including gtfB, ftf, gbpB, relA, brpA, smu630, comDE, vicR were increased (6.10 ± 0.12, 3.34 ± 0.07, 8.75 ± 0.13, 2.96 ± 0.04, 5.20 ± 0.19, 2.20 ± 0.06, 2.32 ± 0.07 and 10.67 ± 0.57 fold) compared to the wild-type strain (P < 0.05). Quorum sensing luxS gene can promote the biofilm formation of Sm.

Systematic Investigation of Expression of G2/M Transition Genes Reveals CDC25 Alteration in Nonfunctioning Pituitary Adenomas.

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Butz, Henriett; Németh, Kinga; Czenke, Dóra; Likó, István; Czirják, Sándor; Zivkovic, Vladimir; Baghy, Kornélia; Korbonits, Márta; Kovalszky, Ilona; Igaz, Péter; Rácz, Károly; Patócs, Attila

2017-07-01

Dysregulation of G1/S checkpoint of cell cycle has been reported in pituitary adenomas. In addition, our previous finding showing that deregulation of Wee1 kinase by microRNAs together with other studies demonstrating alteration of G2/M transition in nonfunctioning pituitary adenomas (NFPAs) suggest that G2/M transition may also be important in pituitary tumorigenesis. To systematically study the expression of members of the G2/M transition in NFPAs and to investigate potential microRNA (miRNA) involvement. Totally, 80 NFPA and 14 normal pituitary (NP) tissues were examined. Expression of 46 genes encoding members of the G2/M transition was profiled on 34 NFPA and 10 NP samples on TaqMan Low Density Array. Expression of CDC25A and two miRNAs targeting CDC25A were validated by individual quantitative real time PCR using TaqMan assays. Protein expression of CDC25A, CDC25C, CDK1 and phospho-CDK1 (Tyr-15) was investigated on tissue microarray and immunohistochemistry. Several genes' expression alteration were observed in NFPA compared to normal tissues by transcription profiling. On protein level CDC25A and both the total and the phospho-CDK1 were overexpressed in adenoma tissues. CDC25A correlated with nuclear localized CDK1 (nCDK1) and with tumor size and nCDK1 with Ki-67 index. Comparing primary vs. recurrent adenomas we found that Ki-67 proliferation index was higher and phospho-CDK1 (inactive form) was downregulated in recurrent tumors compared to primary adenomas. Investigating the potential causes behind CDC25A overexpression we could not find copy number variation at the coding region nor expression alteration of CDC25A regulating transcription factors however CDC25A targeting miRNAs were downregulated in NFPA and negatively correlated with CDC25A expression. Our results suggest that among alterations of G2/M transition of the cell cycle, overexpression of the CDK1 and CDC25A may have a role in the pathogenesis of the NFPA and that CDC25A is potentially

Overexpression of SbMyb60 impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in sorghum bicolor

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The phenylpropanoid biosynthesis pathway that generates lignin subunits represents a significant target to alter the abundance and composition of lignin. The major regulators of phenylpropanoid metabolism are myb transcription factors, which have been shown to modulate secondary cell wall compositi...

Role of neuropsin in parvalbumin immunoreactivity changes in hippocampal basket terminals of mice reared in various environments

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Harumitsu eSuzuki

2014-12-01

Full Text Available In vitro approaches have suggested that neuropsin (or kallikrein 8/KLK8, which controls gamma-aminobutyric acid (GABA neurotransmission through neuregulin-1 and its receptor (ErbB4, is involved in neural plasticity (Tamura et al., 2012, 2013. In the present study, we examined whether parvalbumin (PV-positive neuronal networks, the majority of which are ErbB4-positive GABAergic interneurons, are controlled by neuropsin in tranquil and stimulated voluntarily behaving mice.PV-immunoreactive fibers surrounding hippocampal pyramidal and granular neurons in mice reared in their home cage were decreased in neuropsin-deficient mice, suggesting that neuropsin controls PV immunoreactivity. One- or two-week exposures of wild mice to novel environments, in which they could behave freely and run voluntarily in a wheel resulted in a marked upregulation of both neuropsin mRNA and protein in the hippocampus. To elucidate the functional relevance of the increase in neuropsin during exposure to a rich environment, the intensities of PV-immunoreactive fibers were compared between neuropsin-deficient and wild-type mice under environmental stimuli. When mice were transferred into novel cages (large cages with toys, the intensity of PV-immunoreactive fibers increased in wild-type mice and neuropsin-deficient mice. Therefore, behavioral stimuli control a neuropsin-independent form of PV immunoreactivity. However, the neuropsin-dependent part of the change in PV-immunoreactive fibers may occur in the stimulated hippocampus because increased levels of neuropsin continued during these enriched conditions.

A53T-alpha-synuclein overexpression impairs dopamine signaling and striatal synaptic plasticity in old mice.

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Alexander Kurz

2010-07-01

Full Text Available Parkinson's disease (PD, the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression or the A53T missense mutation of the presynaptic protein alpha-synuclein (SNCA. PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons.Here, we used two mouse lines overexpressing human A53T-SNCA and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. To characterize the progression, we employed young adult as well as old mice. Analysis of striatal neurotransmitter content demonstrated that dopamine (DA levels correlated directly with the level of expression of SNCA, an observation also made in SNCA-deficient (knockout, KO mice. However, the elevated DA levels in the striatum of old A53T-SNCA overexpressing mice may not be transmitted appropriately, in view of three observations. First, a transcriptional downregulation of the extraneural DA degradation enzyme catechol-ortho-methytransferase (COMT was found. Second, an upregulation of DA receptors was detected by immunoblots and autoradiography. Third, extensive transcriptome studies via microarrays and quantitative real-time RT-PCR (qPCR of altered transcript levels of the DA-inducible genes Atf2, Cb1, Freq, Homer1 and Pde7b indicated a progressive and genotype-dependent reduction in the postsynaptic DA response. As a functional consequence, long term depression (LTD was absent in corticostriatal slices from old transgenic mice.Taken together, the dysfunctional neurotransmission and impaired synaptic plasticity seen in the A53T-SNCA overexpressing mice reflect early changes within the basal ganglia prior to frank neurodegeneration. As a model of preclinical stages of PD, such insights may help to develop neuroprotective therapeutic approaches.

Immunohistochemical determination of HER-2/neu overexpression in malignant melanoma reveals no prognostic value, while c-Kit (CD117 overexpression exhibits potential therapeutic implications

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Potti Anil

2003-01-01

Full Text Available Abstract Background HER-2/neu and c-kit (CD117 onco-protein are increasingly being recognized as targets for therapy in solid tumors, but data on their role in malignant melanoma is currently limited. We studied the prevalence of overexpression of HER-2/neu and c-Kit in 202 patients with malignant melanoma to evaluate a possible prognostic value of these molecular targets in malignant melanoma. Methods Overexpression of HER-2/neu and c-Kit was evaluated using immunohistochemical assays in 202 archival tissue specimens. Results Between 1991 and 2001, 202 subjects (109 males; 54% and 93 females; 46% with malignant melanoma were studied with a mean age of 57 years (age range: 15–101 years. The most common histologic type was amelanotic melanoma (n = 62; 30.7% followed by superficial spreading melanoma (n = 54; 26.7%. The depth of penetration of melanoma (Breslow thickness, pT Stage ranged from 0.4 mm (stage pT1 to 8.0 mm (stage pT4A. Mean thickness was 2.6 mm (stage pT3A. The ECOG performance scores ranged from 0 to 3. Only 2 patients (0.9% revealed HER-2/neu overexpression, whereas 46 (22.8% revealed c-Kit overexpression. Multivariate analysis performed did not show a significant difference in survival between c-Kit positive and negative groups (p = 0.36. Interestingly, not only was c-Kit more likely to be overexpressed in the superficial spreading type, a preliminary association between the presence or absence of c-Kit overexpression and the existence of another second primary tumor was also observed. Conclusions The results of our large study indicate that the HER-2/neu onco-protein neither has a role in melanogenesis nor is a potential target for clinical trials with monoclonal antibody therapy. This indicates there is no role for its testing in patients with malignant melanoma. Although c-Kit, expressed preferentially in the superficial spreading type, may not have prognostic value, it does have significant therapeutic implications as a

Ectopic overexpression of WsSGTL1, a sterol glucosyltransferase gene in Withania somnifera, promotes growth, enhances glycowithanolide and provides tolerance to abiotic and biotic stresses.

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Saema, Syed; Rahman, Laiq Ur; Singh, Ruchi; Niranjan, Abhishek; Ahmad, Iffat Zareen; Misra, Pratibha

2016-01-01

Overexpression of sterol glycosyltransferase (SGTL1) gene of Withania somnifera showing its involvement in glycosylation of withanolide that leads to enhanced growth and tolerance to biotic and abiotic stresses. Withania somnifera is widely used in Ayurvedic medicines for over 3000 years due to its therapeutic properties. It contains a variety of glycosylated steroids called withanosides that possess neuroregenerative, adaptogenic, anticonvulsant, immunomodulatory and antioxidant activities. The WsSGTL1 gene specific for 3β-hydroxy position has a catalytic specificity to glycosylate withanolide and sterols. Glycosylation not only stabilizes the products but also alters their physiological activities and governs intracellular distribution. To understand the functional significance and potential of WsSGTL1 gene, transgenics of W. somnifera were generated using Agrobacterium tumefaciens-mediated transformation. Stable integration and overexpression of WsSGTL1 gene were confirmed by Southern blot analysis followed by quantitative real-time PCR. The WsGTL1 transgenic plants displayed number of alterations at phenotypic and metabolic level in comparison to wild-type plants which include: (1) early and enhanced growth with leaf expansion and increase in number of stomata; (2) increased production of glycowithanolide (majorly withanoside V) and campesterol, stigmasterol and sitosterol in glycosylated forms with reduced accumulation of withanolides (withaferin A, withanolide A and withanone); (3) tolerance towards biotic stress (100 % mortality of Spodoptera litura), improved survival capacity under abiotic stress (cold stress) and; (4) enhanced recovery capacity after cold stress, as indicated by better photosynthesis performance, chlorophyll, anthocyanin content and better quenching regulation of PSI and PSII. Our data demonstrate overexpression of WsSGTL1 gene which is responsible for increase in glycosylated withanolide and sterols, and confers better growth and

Clinicopathological correlation and prognostic significance of sonic hedgehog protein overexpression in human gastric cancer.

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Niu, Yanyang; Li, Fang; Tang, Bo; Shi, Yan; Hao, Yingxue; Yu, Peiwu

2014-01-01

This study investigated the expression of Sonic Hedgehog (Shh) protein in gastric cancer, and correlated it with clinicopathological parameters. The prognostic significance of Shh protein was analyzed. Shh protein expression was evaluated in 113 cases of gastric cancer and 60 cases of normal gastric mucosa. The immunoreactivity was scored semi quantitatively as: 0 = absent; 1 = weak; 2 = moderate; and 3 = strong. All cases were further classified into two groups, namely non-overexpression group with score 0 or 1, and overexpression group with score 2 or 3. The overexpression of Shh protein was correlated with clinicopathological parameters. Survival analysis was then performed to determine the Shh protein prognostic significance in gastric cancer. In immunohistochemistry study, nineteen (31.7%) normal gastric mucosa revealed Shh protein overexpression, while eighty-one (71.7%) gastric cancer revealed overexpression. The expression of Shh protein were significantly higher in gastric cancer tissues than in normal gastric mucosa (P overexpression and non-expression groups P = 0.168 and 0.071). However, Shh overexpression emerged as a significant independent prognostic factor in multivariate Cox regression analysis (hazard ratio 1.187, P = 0.041). Shh protein expression is upregulated and is statistically correlated with age, tumor differentiation, depth of invasion, pathologic staging, and nodal metastasis. The Shh protein overexpression is a significant independent prognostic factor in multivariate Cox regression analysis in gastric cancer.

Extending the Impact of RAC1b Overexpression to Follicular Thyroid Carcinomas

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Márcia Faria

2016-01-01

Full Text Available RAC1b is a hyperactive variant of the small GTPase RAC1 known to be a relevant molecular player in different cancers. Previous studies from our group lead to the evidence that its overexpression in papillary thyroid carcinoma (PTC is associated with an unfavorable prognosis. In the present study, we intended to extend the analysis of RAC1b expression to thyroid follicular neoplasms and to seek for clinical correlations. RAC1b expression levels were determined by RT-qPCR in thyroid follicular tumor samples comprising 23 follicular thyroid carcinomas (FTCs and 33 follicular thyroid adenomas (FTAs. RAC1b was found to be overexpressed in 33% of carcinomas while no RAC1b overexpression was documented among follicular adenomas. Patients with a diagnosis of FTC were divided into two groups based on longitudinal evolution and final outcome. RAC1b overexpression was significantly associated with both the presence of distant metastases (P = 0.01 and poorer clinical outcome (P = 0.01 suggesting that, similarly to that previously found in PTCs, RAC1b overexpression in FTCs is also associated with worse outcomes. Furthermore, the absence of RAC1b overexpression in follicular adenomas hints its potential as a molecular marker likely to contribute, in conjunction with other putative markers, to the preoperative differential diagnosis of thyroid follicular lesions.

Extending the Impact of RAC1b Overexpression to Follicular Thyroid Carcinomas

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Faria, Márcia; Capinha, Liliana; Simões-Pereira, Joana; Bugalho, Maria João; Silva, Ana Luísa

2016-01-01

RAC1b is a hyperactive variant of the small GTPase RAC1 known to be a relevant molecular player in different cancers. Previous studies from our group lead to the evidence that its overexpression in papillary thyroid carcinoma (PTC) is associated with an unfavorable prognosis. In the present study, we intended to extend the analysis of RAC1b expression to thyroid follicular neoplasms and to seek for clinical correlations. RAC1b expression levels were determined by RT-qPCR in thyroid follicular tumor samples comprising 23 follicular thyroid carcinomas (FTCs) and 33 follicular thyroid adenomas (FTAs). RAC1b was found to be overexpressed in 33% of carcinomas while no RAC1b overexpression was documented among follicular adenomas. Patients with a diagnosis of FTC were divided into two groups based on longitudinal evolution and final outcome. RAC1b overexpression was significantly associated with both the presence of distant metastases (P = 0.01) and poorer clinical outcome (P = 0.01) suggesting that, similarly to that previously found in PTCs, RAC1b overexpression in FTCs is also associated with worse outcomes. Furthermore, the absence of RAC1b overexpression in follicular adenomas hints its potential as a molecular marker likely to contribute, in conjunction with other putative markers, to the preoperative differential diagnosis of thyroid follicular lesions. PMID:27127508

Sensory Deprivation during Early Postnatal Period Alters the Density of Interneurons in the Mouse Prefrontal Cortex

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Hiroshi Ueno

2015-01-01

Full Text Available Early loss of one sensory system can cause improved function of other sensory systems. However, both the time course and neuronal mechanism of cross-modal plasticity remain elusive. Recent study using functional MRI in humans suggests a role of the prefrontal cortex (PFC in cross-modal plasticity. Since this phenomenon is assumed to be associated with altered GABAergic inhibition in the PFC, we have tested the hypothesis that early postnatal sensory deprivation causes the changes of inhibitory neuronal circuit in different regions of the PFC of the mice. We determined the effects of sensory deprivation from birth to postnatal day 28 (P28 or P58 on the density of parvalbumin (PV, calbindin (CB, and calretinin (CR neurons in the prelimbic, infralimbic, and dorsal anterior cingulate cortices. The density of PV and CB neurons was significantly increased in layer 5/6 (L5/6. Moreover, the density of CR neurons was higher in L2/3 in sensory deprived mice compared to intact mice. These changes were more prominent at P56 than at P28. These results suggest that long-term sensory deprivation causes the changes of intracortical inhibitory networks in the PFC and the changes of inhibitory networks in the PFC may contribute to cross-modal plasticity.

The Transcription Cofactor Swi6 of the Fusarium graminearum Is Involved in Fusarium Graminearum Virus 1 Infection-Induced Phenotypic Alterations

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Moonil Son

2016-08-01

Full Text Available The transcription cofactor Swi6 plays important roles in regulating vegetative growth and meiosis in Saccharomyces cerevisiae. Functions of Swi6 ortholog were also characterized in Fusarium graminearum which is one of the devastating plant pathogenic fungi. Here, we report possible role of FgSwi6 in the interaction between F. graminearum and Fusarium graminearum virus 1 (FgV1 strain DK21. FgV1 perturbs biological characteristics of host fungi such as vegetative growth, sporulation, pigmentation, and reduction of the virulence (hypovirulence of its fungal host. To characterize function(s of FgSWI6 gene during FgV1 infection, targeted deletion, over-expression, and complementation mutants were generated and further infected successfully with FgV1. Deletion of FgSwi6 led to severe reduction of vegetative growth even aerial mycelia while over-expression did not affect any remarkable alteration of phenotype in virus-free isolates. Virus-infected (VI FgSWI6 deletion isolate exhibited completely delayed vegetative growth. However, VI FgSWI6 over-expression mutant grew faster than any other VI isolates. To verify whether these different growth patterns in VI isolates, viral RNA quantification was carried out using qRT-PCR. Surprisingly, viral RNA accumulations in VI isolates were similar regardless of introduced mutations. These results provide evidence that FgSWI6 might play important role(s in FgV1 induced phenotype alteration such as delayed vegetative growth.

Correlation between human papillomavirus and p16 overexpression in oropharyngeal tumours

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Grønhøj Larsen, C; Gyldenløve, M; Jensen, D H

2014-01-01

A significant proportion of squamous cell carcinomas of the oropharynx (OP-SCC) are related to human papillomavirus (HPV) infection and p16 overexpression. This subgroup proves better prognosis and survival but no evidence exists on the correlation between HPV and p16 overexpression based on diag...

Transgenic overexpression of adenine nucleotide translocase 1 protects ischemic hearts against oxidative stress.

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Klumpe, Inga; Savvatis, Konstantinos; Westermann, Dirk; Tschöpe, Carsten; Rauch, Ursula; Landmesser, Ulf; Schultheiss, Heinz-Peter; Dörner, Andrea

2016-06-01

Ischemia impairs the adenine nucleotide translocase (ANT), which transports ADP and ATP across the inner mitochondrial membrane. We investigated whether ANT1 overexpression has protective effects on ischemic hearts. Myocardial infarction was induced in wild-type (WT) and heart-specific ANT1-transgenic (ANT1-TG) rats, and hypoxia was set in isolated cardiomyocytes. ANT1 overexpression reduced the myocardial infarct area and increased the survival rate of infarcted rats. Reduced ANT1 expression and increased 4-hydroxynonenal modification of ANT paralleled to impaired ANT function in infarcted WT hearts. ANT1 overexpression improved ANT expression and function. This was accompanied by reduced mitochondrial cytochrome C release and caspase-3 activation. ANT1-TG hearts suffered less from oxidative stress, as shown by lower protein carbonylation and 4-hydroxynonenal modification of ANT. ANT1 overexpression also increased cell survival of hypoxic cardiomyocytes and attenuated reactive oxygen species (ROS) production. This was linked to higher stability of mitochondrial membrane potential and lower activity of ROS detoxifying catalase. ANT1-TG cardiomyocytes also showed higher resistance against H2O2 treatment, which was independent of catalase activity. In conclusion, ANT1 overexpression compensates impaired ANT activity under oxygen-restricted conditions. It reduces ROS production and oxidative stress, stabilizes mitochondrial integrity, and increases survival, making ANT1 a component in ROS management and heart protection during ischemia. ANT1 overexpression reduces infarct size and increases survival after infarction. ANT1 overexpression compensates restricted ANT expression and function in infarcted hearts. Increased ANT1 expression enhances mitochondrial integrity. ANT1-overexpressing hearts reduce oxidative stress by decreasing ROS generation. ANT1 is a component in ROS management and heart protection.

Overexpression of K-p21Ras play a prominent role in lung cancer

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Zhang, Peng-bo; Zhou, Xin-liang; Yang, Ju-lun

2018-06-01

The proto-oncogene ras product, p21Ras, has been found overexpression in many human tumors. However, the subtypes of overexpressed p21Ras still remain unclear. The purpose of this study was to investigate overexpressed isoforms of p21Ras and their roles in the progress of lung cancer. Method: The expression of total p21Ras in normal lung tissues and lung cancers was determined by immunohistochemically staining with monoclonal antibody (Mab) KGHR-1 which could recognize and broad spectrum reaction with the (K/H/N) ras protein. Then, the isoforms of p21Ras was examined by specific Mab for each p21Ras subtypes. Results: Low expression of total p21Ras was found in 26.67% (8/30) of normal lung tissues, and 81.31% (87/107) of adenocarcinoma harbored overexpressed total p21Ras. Besides, 70.00% (35/50) of squamous cell carcinoma were detected overexpressed total p21Ras. In addition, 122 lung cancer tissues from overexpression of total p21Ras protein were selected to detect the expression of each subtype. And all the 122 lung cancer tissues were K-p21Ras overexpression. Moreover, there was a statistical significance difference between the expression level of total p21Ras and differentiation, and the same results were observed between the expression level of total p21Ras and lymph node metastasis (P0.05). Conclusions: Overexpression of K-p21Ras plays a prominent role in the progress of lung cancer and it is suggested that the p21Ras could serve as a promising treatment target in lung cancer.

Overexpression of MDM2 protein in ameloblastomas as compared to adenomatoid odontogenic tumor

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A Krishna

2012-01-01

Full Text Available Background: Recent studies on odontogenic tumors have identified various molecular alterations responsible for their development, and determination of epithelial proliferation is a useful means of investigating the differences in biologic behavior of these tumors. One such specific marker to identify proliferative activity and tumor aggressiveness by immunohistochemistry (IHC is MDM2, 90-95kDa protein. Objective: This immunohistochemical study using MDM2 expression was undertaken to understand better the diverse biological activity of two groups of odontogenic tumors namely ameloblastoma and adenomatoid odontogenic tumor (AOT based on their cell proliferation activity. Materials and Methods: A total of 50 cases, comprising of 36 ameloblastoma samples and 14 AOT samples, were subjected to heat-induced antigen retrieval method using citrate buffer in a pressure cooker. Consequently, the sections were stained with MDM2 monoclonal antibody and visualized using an LSAB+ kit. Results: In ameloblastomas, statistically significant association was seen between plexiform ameloblastomas, follicular ameloblastomas with granular cell changes, desmoplastic and unicystic variants. The predominant nuclear staining by MDM2 revealed overexpression in ameloblastomas as compared to AOT. Conclusion: The MDM2 overexpression noticed in plexiform ameloblastoma, follicular ameloblastoma with granular cell changes and acanthomatous ameloblastoma when compared to simple unicystic and desmoplastic ameloblastoma suggest a relatively enhanced proliferative phenotype of these solid multicystic variants of ameloblastomas. On overall comparison, higher expression was noted in ameloblastomas when compared to AOT. This indicates differences in the aggressive nature between these two groups of odontogenic tumors favoring the perception of a greater aggressive nature of ameloblastomas.

Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer

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Duffy, Supipi; Fam, Hok Khim; Wang, Yi Kan; Styles, Erin B.; Kim, Jung-Hyun; Ang, J. Sidney; Singh, Tejomayee; Larionov, Vladimir; Shah, Sohrab P.; Andrews, Brenda; Boerkoel, Cornelius F.; Hieter, Philip

2016-01-01

Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1. Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors. PMID:27551064

The production of nitric oxide in EL4 lymphoma cells overexpressing growth hormone.

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Arnold, Robyn E; Weigent, Douglas A

2003-01-01

Growth hormone (GH) is produced by immunocompetent cells and has been implicated in the regulation of a multiplicity of functions in the immune system involved in growth and activation. However, the actions of endogenous or lymphocyte GH and its contribution to immune reactivity when compared with those of serum or exogenous GH are still unclear. In the present study, we overexpressed lymphocyte GH in EL4 lymphoma cells, which lack the GH receptor (GHR), to determine the role of endogenous GH in nitric oxide (NO) production and response to genotoxic stress. Western blot analysis demonstrated that the levels of GH increased approximately 40% in cells overexpressing GH (GHo) when compared with cells with vector alone. The results also show a substantial increase in NO production in cells overexpressing GH that could be blocked by N(G)-monomethyl-L-arginine (L-NMMA), an L-arginine analogue that competitively inhibits all three isoforms of nitric oxide synthase (NOS). No evidence was obtained to support an increase in peroxynitrite in cells overexpressing GH. Overexpression of GH increased NOS activity, inducible nitric oxide synthase (iNOS) promoter activity, and iNOS protein expression, whereas endothelial nitric oxide synthase and neuronal nitric oxide synthase protein levels were essentially unchanged. In addition, cells overexpressing GH showed increased arginine transport ability and intracellular arginase activity when compared with control cells. GH overexpression appeared to protect cells from the toxic effects of the DNA alkylating agent methyl methanesulfonate. This possibility was suggested by maintenance of the mitochondrial transmembrane potential in cells overexpressing GH when compared with control cells that could be blocked by L-NMMA. Taken together, the data support the notion that lymphocyte GH, independently of the GH receptor, may play a key role in the survival of lymphocytes exposed to stressful stimuli via the production of NO.

Functional and gene expression analysis of hTERT overexpressed endothelial cells

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Haruna Takano

2008-09-01

Full Text Available Haruna Takano1, Satoshi Murasawa1,2, Takayuki Asahara1,2,31Institute of Biomedical Research and Innovation, Kobe, Japan; 2RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; 3Tokai University of School of Medicine, Tokai, JapanAbstract: Telomerase dysfunction contributes to cellular senescence. Recent advances indicate the importance of senescence in maintaining vascular cell function in vitro. Human telomerase reverse transcriptase (hTERT overexpression is thought to lead to resistance to apoptosis and oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to generate an immortal endothelial cell line from human umbilical vein endothelial cells using a no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial cell senescence in vitro. High levels of hTERT genes and endothelial cell-specific markers were expressed during long-term culture. Also, angiogenic responses were observed in hTERT overexpressed endothelial cell. These cells showed a delay in senescence and appeared more resistant to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might contribute to anti-apoptosis and survival effects in endothelial lineage cells.Keywords: endothelial, telomerase, senescence, oxidative stress, anti-apoptosis, PI3K/Akt pathway

Targeted Transgenic Overexpression of Mitochondrial Thymidine Kinase (TK2) Alters Mitochondrial DNA (mtDNA) and Mitochondrial Polypeptide Abundance : Transgenic TK2, mtDNA, and Antiretrovirals

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Hosseini, Seyed H.; Kohler, James J.; Haase, Chad P.; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-01-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-γ. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK...

Aurora-A overexpression and aneuploidy predict poor outcome in serous ovarian carcinoma.

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Lassus, Heini; Staff, Synnöve; Leminen, Arto; Isola, Jorma; Butzow, Ralf

2011-01-01

Aurora-A is a potential oncogene and therapeutic target in ovarian carcinoma. It is involved in mitotic events and overexpression leads to centrosome amplification and chromosomal instability. The objective of this study was to evaluate the clinical significance of Aurora-A and DNA ploidy in serous ovarian carcinoma. Serous ovarian carcinomas were analysed for Aurora-A protein by immunohistochemistry (n=592), Aurora-A copy number by CISH (n=169), Aurora-A mRNA by real-time PCR (n=158) and DNA ploidy by flowcytometry (n=440). Overexpression of Aurora-A was found in 27% of the tumors, cytoplasmic overexpression in 11% and nuclear in 17%. The cytoplasmic and nuclear overexpression were nearly mutually exclusive. Both cytoplasmic and nuclear overexpression were associated with shorter survival, high grade, high proliferation index and aberrant p53. Interestingly, only cytoplasmic expression was associated with aneuploidy and expression of phosphorylated Aurora-A. DNA ploidy was associated with poor patient outcome as well as aggressive clinicopathological parameters. In multivariate analysis, Aurora-A overexpression appeared as an independent prognostic factor for disease-free survival, together with grade, stage and ploidy. Aurora-A protein expression is strongly linked with poor patient outcome and aggressive disease characteristics, which makes Aurora-A a promising biomarker and a potential therapeutic target in ovarian carcinoma. Cytoplasmic and nuclear Aurora-A protein may have different functions. DNA aneuploidy is a strong predictor of poor prognosis in serous ovarian carcinoma. Copyright © 2010 Elsevier Inc. All rights reserved.

Parvalbumin-expressing interneurons can act solo while somatostatin-expressing interneurons act in chorus in most cases on cortical pyramidal cells.

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Safari, Mir-Shahram; Mirnajafi-Zadeh, Javad; Hioki, Hiroyuki; Tsumoto, Tadaharu

2017-10-06

Neural circuits in the cerebral cortex consist primarily of excitatory pyramidal (Pyr) cells and inhibitory interneurons. Interneurons are divided into several subtypes, in which the two major groups are those expressing parvalbumin (PV) or somatostatin (SOM). These subtypes of interneurons are reported to play distinct roles in tuning and/or gain of visual response of pyramidal cells in the visual cortex. It remains unclear whether there is any quantitative and functional difference between the PV → Pyr and SOM → Pyr connections. We compared unitary inhibitory postsynaptic currents (uIPSCs) evoked by electrophysiological activation of single presynaptic interneurons with population IPSCs evoked by photo-activation of a mass of interneurons in vivo and in vitro in transgenic mice in which PV or SOM neurons expressed channelrhodopsin-2, and found that at least about 14 PV neurons made strong connections with a postsynaptic Pyr cell while a much larger number of SOM neurons made weak connections. Activation or suppression of single PV neurons modified visual responses of postsynaptic Pyr cells in 6 of 7 pairs whereas that of single SOM neurons showed no significant modification in 8 of 11 pairs, suggesting that PV neurons can act solo whereas most of SOM neurons may act in chorus on Pyr cells.

Over-expression of HER-2 is associated with the stage in ...

African Journals Online (AJOL)

Background: The frequency of over-expression of human epidermal growth factor receptor-2 (HER-2) in bladder cancer is one of the highest among all human malignancies. This over-expression is thought to play a role in aberrant proliferation of cancer cells. Studies on HER-2 expression in bladder carcinoma have shown ...

l-Proline, GABA Synthesis and Gamma Oscillations in Schizophrenia.

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Volk, David W; Gonzalez-Burgos, Guillermo; Lewis, David A

2016-12-01

Altered inhibition from parvalbumin-containing GABA neurons is thought to contribute to impaired gamma frequency oscillations and cognitive deficits in schizophrenia. Crabtree and colleagues report that proline dehydrogenase deficits produce excessive cytosolic levels of the GABA-mimetic l-proline which impairs GABA synthesis and gamma oscillations in a manner that mimics schizophrenia. Copyright © 2016 Elsevier Ltd. All rights reserved.

CMV-Promoter Driven Codon-Optimized Expression Alters the Assembly Type and Morphology of a Reconstituted HERV-K(HML-2

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Oliver Hohn

2014-11-01

Full Text Available The HERV-K(HML-2 family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations.

CMV-promoter driven codon-optimized expression alters the assembly type and morphology of a reconstituted HERV-K(HML-2).

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Hohn, Oliver; Hanke, Kirsten; Lausch, Veronika; Zimmermann, Anja; Mostafa, Saeed; Bannert, Norbert

2014-11-11

The HERV-K(HML-2) family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM) and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations.

Development of a hypoallergenic recombinant parvalbumin for first-in-man subcutaneous immunotherapy of fish allergy.

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Zuidmeer-Jongejan, Laurian; Huber, Hans; Swoboda, Ines; Rigby, Neil; Versteeg, Serge A; Jensen, Bettina M; Quaak, Suzanne; Akkerdaas, Jaap H; Blom, Lars; Asturias, Juan; Bindslev-Jensen, Carsten; Bernardi, Maria L; Clausen, Michael; Ferrara, Rosa; Hauer, Martina; Heyse, Jet; Kopp, Stephan; Kowalski, Marek L; Lewandowska-Polak, Anna; Linhart, Birgit; Maderegger, Bernhard; Maillere, Bernard; Mari, Adriano; Martinez, Alberto; Mills, E N Clare; Neubauer, Angela; Nicoletti, Claudio; Papadopoulos, Nikolaos G; Portoles, Antonio; Ranta-Panula, Ville; Santos-Magadan, Sara; Schnoor, Heidi J; Sigurdardottir, Sigurveig T; Stahl-Skov, Per; Stavroulakis, George; Stegfellner, Georg; Vázquez-Cortés, Sonia; Witten, Marianne; Stolz, Frank; Poulsen, Lars K; Fernandez-Rivas, Montserrat; Valenta, Rudolf; van Ree, Ronald

2015-01-01

The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product. Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability. The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine. © 2015 S. Karger AG, Basel.

Long-term polarization of microglia upon alpha-synuclein overexpression in nonhuman primates

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Barkholt, Pernille; Sanchez-Guajardo, Vanesa Maria; Kirik, Denis

2012-01-01

We have previously shown that persistent ﰇ-sy- nuclein overexpression in ventral midbrain of marmoset leads to a distinctive neurodegenerative process and motor defects. The neurodegeneration was confined to caudate putamen dopaminergic fibers in animals overexpressing wild-type (wt) ﰇ-synuclein....

Effects of striatal ΔFosB overexpression and ketamine on social defeat stress-induced anhedonia in mice.

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Donahue, Rachel J; Muschamp, John W; Russo, Scott J; Nestler, Eric J; Carlezon, William A

2014-10-01

Chronic social defeat stress (CSDS) produces persistent behavioral adaptations in mice. In many behavioral assays, it can be difficult to determine if these adaptations reflect core signs of depression. We designed studies to characterize the effects of CSDS on sensitivity to reward because anhedonia (reduced sensitivity to reward) is a defining characteristic of depressive disorders in humans. We also examined the effects of striatal ΔFosB overexpression and the N-methyl-D-aspartate receptor antagonist ketamine, both of which promote resilience, on CSDS-induced alterations in reward function and social interaction. Intracranial self-stimulation (ICSS) was used to quantify CSDS-induced changes in reward function. Mice were implanted with lateral hypothalamic electrodes, and ICSS thresholds were measured after each of 10 daily CSDS sessions and during a 5-day recovery period. We also examined if acute intraperitoneal administration of ketamine (2.5-20 mg/kg) reverses CSDS-induced effects on reward or, in separate mice, social interaction. ICSS thresholds were increased by CSDS, indicating decreases in the rewarding impact of lateral hypothalamic stimulation (anhedonia). This effect was attenuated in mice overexpressing ∆FosB in striatum, consistent with pro-resilient actions of this transcription factor. High, but not low, doses of ketamine administered after completion of the CSDS regimen attenuated social avoidance in defeated mice, although this effect was transient. Ketamine did not block CSDS-induced anhedonia in the ICSS test. This study found that CSDS triggers persistent anhedonia and confirms that ΔFosB overexpression produces stress resilience. The findings of this study also indicate that acute administration of ketamine fails to attenuate CSDS-induced anhedonia despite reducing other depression-related behavioral abnormalities. Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Twinkle overexpression prevents cardiac rupture after myocardial infarction by alleviating impaired mitochondrial biogenesis.

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Inoue, Takahiro; Ikeda, Masataka; Ide, Tomomi; Fujino, Takeo; Matsuo, Yuka; Arai, Shinobu; Saku, Keita; Sunagawa, Kenji

2016-09-01

Cardiac rupture is a fatal complication after myocardial infarction (MI). However, the detailed mechanism underlying cardiac rupture after MI remains to be fully elucidated. In this study, we investigated the role of mitochondrial DNA (mtDNA) and mitochondria in the pathophysiology of cardiac rupture by analyzing Twinkle helicase overexpression mice (TW mice). Twinkle overexpression increased mtDNA copy number approximately twofold and ameliorated ischemic cardiomyopathy at day 28 after MI. Notably, Twinkle overexpression markedly prevented cardiac rupture and improved post-MI survival, accompanied by the suppression of MMP-2 and MMP-9 in the MI border area at day 5 after MI when cardiac rupture frequently occurs. Additionally, these cardioprotective effects of Twinkle overexpression were abolished in transgenic mice overexpressing mutant Twinkle with an in-frame duplication of amino acids 353-365, which resulted in no increases in mtDNA copy number. Furthermore, although apoptosis and oxidative stress were induced and mitochondria were damaged in the border area, these injuries were improved in TW mice. Further analysis revealed that mitochondrial biogenesis, including mtDNA copy number, transcription, and translation, was severely impaired in the border area at day 5 In contrast, Twinkle overexpression maintained mtDNA copy number and restored the impaired transcription and translation of mtDNA in the border area. These results demonstrated that Twinkle overexpression alleviated impaired mitochondrial biogenesis in the border area through maintained mtDNA copy number and thereby prevented cardiac rupture accompanied by the reduction of apoptosis and oxidative stress, and suppression of MMP activity. Copyright © 2016 the American Physiological Society.

A novel vector-based method for exclusive overexpression of star-form microRNAs.

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Bo Qu

Full Text Available The roles of microRNAs (miRNAs as important regulators of gene expression have been studied intensively. Although most of these investigations have involved the highly expressed form of the two mature miRNA species, increasing evidence points to essential roles for star-form microRNAs (miRNA*, which are usually expressed at much lower levels. Owing to the nature of miRNA biogenesis, it is challenging to use plasmids containing miRNA coding sequences for gain-of-function experiments concerning the roles of microRNA* species. Synthetic microRNA mimics could introduce specific miRNA* species into cells, but this transient overexpression system has many shortcomings. Here, we report that specific miRNA* species can be overexpressed by introducing artificially designed stem-loop sequences into short hairpin RNA (shRNA overexpression vectors. By our prototypic plasmid, designed to overexpress hsa-miR-146b-3p, we successfully expressed high levels of hsa-miR-146b-3p without detectable change of hsa-miR-146b-5p. Functional analysis involving luciferase reporter assays showed that, like natural miRNAs, the overexpressed hsa-miR-146b-3p inhibited target gene expression by 3'UTR seed pairing. Our demonstration that this method could overexpress two other miRNAs suggests that the approach should be broadly applicable. Our novel strategy opens the way for exclusively stable overexpression of miRNA* species and analyzing their unique functions both in vitro and in vivo.

Localization of peroxisome proliferator-activated receptor alpha (PPARα) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) in cells expressing the Ca2+-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus

Science.gov (United States)

Rivera, Patricia; Arrabal, Sergio; Vargas, Antonio; Blanco, Eduardo; Serrano, Antonia; Pavón, Francisco J.; Rodríguez de Fonseca, Fernando; Suárez, Juan

2014-01-01

The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca2+ fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca2+-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα+/calbindin+ cells were closely surrounded by NAPE-PLD+ fiber varicosities. No pyramidal PPARα+/calbindin+ cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD+/calretinin+ cells were specifically detected in CA3. NAPE-PLD+ puncta surrounded the calretinin+ cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions. PMID:24672435

Co-ordinate regulation of the cytoskeleton in 3T3 cells overexpressing thymosin-beta4.

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Golla, R; Philp, N; Safer, D; Chintapalli, J; Hoffman, R; Collins, L; Nachmias, V T

1997-01-01

In several cell types, short-term increases in the concentration of the G-actin-sequestering peptide thymosin-beta4 (Tbeta4) cause the disassembly of F-actin bundles. To determine the extent of cell adaptability to these reductions in F-actin, we overexpressed Tbeta4 in NIH 3T3 cells. In cell lines with Tbeta4 levels twice those of vector controls, G-actin increased approximately twofold as expected. However, F-actin did not decrease as in short-term experiments but rather also increased approximately twofold so that the G-F ratio remained constant. Surprisingly, the cytoskeletal proteins myosin IIA, alpha-actinin, and tropomyosin also increased nearly twofold. These increases were specific; DNA, total protein, lactic dehydrogenase, profilin, and actin depolymerizing factor levels were unchanged in the overexpressing cells. The Tbeta4 lines spread more fully and adhered to the dish more strongly than vector controls; this altered phenotype correlated with a twofold increase in talin and alpha5-integrin and a nearly threefold increase in vinculin. Focal adhesions, detected by indirect immunofluorescence with antivinculin, were increased in size over the controls. Northern blotting showed that mRNAs for both beta-actin and vinculin were increased twofold in the overexpressing lines. We conclude that 1) NIH 3T3 cells adapt to increased levels of G-actin sequestered by increased Tbeta4 by increasing their total actin so that the F-actin/G-actin ratio remains constant; 2) these cells coordinately increase several cytoskeletal and adhesion plaque proteins; and 3) at least for actin and vinculin, this regulation is at the transcriptional level. We therefore propose that the proteins of this multimember interacting complex making up the actin-based cytoskeleton, are coordinately regulated by factors that control the expression of several proteins. The mechanism may bear similarities to the control of synthesis of another multimember interacting complex, the myofibril of

IL-6 Overexpression in ERG-Positive Prostate Cancer Is Mediated by Prostaglandin Receptor EP2.

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Merz, Constanze; von Mässenhausen, Anne; Queisser, Angela; Vogel, Wenzel; Andrén, Ove; Kirfel, Jutta; Duensing, Stefan; Perner, Sven; Nowak, Michael

2016-04-01

Prostate cancer is the most diagnosed cancer in men and multiple risk factors and genetic alterations have been described. The TMPRSS2-ERG fusion event and the overexpression of the transcription factor ERG are present in approximately 50% of all prostate cancer patients, however, the clinical outcome is still controversial. Prostate tumors produce various soluble factors, including the pleiotropic cytokine IL-6, regulating cellular processes such as proliferation and metastatic segregation. Here, we used prostatectomy samples in a tissue microarray format and analyzed the co-expression and the clinicopathologic data of ERG and IL-6 using immunohistochemical double staining and correlated the read-out with clinicopathologic data. Expression of ERG and IL-6 correlated strongly in prostate tissue samples. Forced expression of ERG in prostate tumor cell lines resulted in significantly increased secretion of IL-6, whereas the down-regulation of ERG decreased IL-6 secretion. By dissecting the underlying mechanism in prostate tumor cell lines we show the ERG-mediated up-regulation of the prostanoid receptors EP2 and EP3. The prostanoid receptor EP2 was overexpressed in human prostate cancer tissue. Furthermore, the proliferation rate and IL-6 secretion in DU145 cells was reduced after treatment with EP2-receptor antagonist. Collectively, our study shows that the expression of ERG in prostate cancer is linked to the expression of IL-6 mediated by the prostanoid receptor EP2. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Overexpression of Insulin-like Growth Factor-1 Receptor Is Associated With Penile Cancer Progression.

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Ball, Mark W; Bezerra, Stephania M; Chaux, Alcides; Faraj, Sheila F; Gonzalez-Roibon, Nilda; Munari, Enrico; Sharma, Rajni; Bivalacqua, Trinity J; Netto, George J; Burnett, Arthur L

2016-06-01

To evaluate insulin-like growth factor-1 receptor (IGF1R) expression in penile cancer and its association with oncologic outcomes. Tissue microarrays were constructed from 53 patients treated at our institution. Expression of IGF1R was evaluated using a Her2-like scoring system. Overexpression was defined as 1+ or greater membranous staining. Association of IGF1R expression with pathologic features was assessed with comparative statistics, and association with local recurrence, progression to nodal or distance metastases, or death was assessed with Kaplan-Meier survival analysis and Cox proportional hazard regression models. Overall, IGF1R overexpression was seen in 33 (62%) cases. With a median follow-up of 27.8 months, IGF1R overexpression was associated with inferior progression-free survival (PFS) (P  =  .003). In a multivariable model controlling for grade, T stage, perineural invasion, and lymphovascular invasion, IGF1R expression was independently associated with disease progression (hazard ratio 2.3, 95% confidence interval 1.1-5.1, P  =  .03. Comparing patients without IGF1R overexpression to those with overexpression, 5-year PFS was 94.1% vs 45.8%. IGF1R overexpression was associated with inferior PFS in penile cancer. Drugs that target IGF1R and downstream messengers may have a therapeutic benefit in patients that exhibit IGF1R overexpression. Copyright © 2016 Elsevier Inc. All rights reserved.

Knockout of NMDA-receptors from parvalbumin interneurons sensitizes to schizophrenia-related deficits induced by MK-801

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Bygrave, A M; Masiulis, S; Nicholson, E; Berkemann, M; Barkus, C; Sprengel, R; Harrison, P J; Kullmann, D M; Bannerman, D M; Kätzel, D

2016-01-01

It has been suggested that a functional deficit in NMDA-receptors (NMDARs) on parvalbumin (PV)-positive interneurons (PV-NMDARs) is central to the pathophysiology of schizophrenia. Supportive evidence come from examination of genetically modified mice where the obligatory NMDAR-subunit GluN1 (also known as NR1) has been deleted from PV interneurons by Cre-mediated knockout of the corresponding gene Grin1 (Grin1ΔPV mice). Notably, such PV-specific GluN1 ablation has been reported to blunt the induction of hyperlocomotion (a surrogate for psychosis) by pharmacological NMDAR blockade with the non-competitive antagonist MK-801. This suggests PV-NMDARs as the site of the psychosis-inducing action of MK-801. In contrast to this hypothesis, we show here that Grin1ΔPV mice are not protected against the effects of MK-801, but are in fact sensitized to many of them. Compared with control animals, Grin1ΔPVmice injected with MK-801 show increased stereotypy and pronounced catalepsy, which confound the locomotor readout. Furthermore, in Grin1ΔPVmice, MK-801 induced medial-prefrontal delta (4 Hz) oscillations, and impaired performance on tests of motor coordination, working memory and sucrose preference, even at lower doses than in wild-type controls. We also found that untreated Grin1ΔPVmice are largely normal across a wide range of cognitive functions, including attention, cognitive flexibility and various forms of short-term memory. Taken together these results argue against PV-specific NMDAR hypofunction as a key starting point of schizophrenia pathophysiology, but support a model where NMDAR hypofunction in multiple cell types contribute to the disease. PMID:27070406

Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants

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Katherine Maringer

2014-08-01

Full Text Available Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA, and dominant negative (DN forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell–cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses.

Alterations of the TP53 Gene in Gastric and Esophageal Carcinogenesis

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Marilanda Ferreira Bellini

2012-01-01

Full Text Available TP53 genes is one of more important tumor suppressor gene, which acts as a potent transcription factor with fundamental role in the maintenance of genetic stability. The development of esophageal and gastric cancers is a multistep process resulting in successive accumulation of genetic alterations that culminates in the malignant transformation. Thus, this study highlights the participation of the main genetic alterations of the TP53 gene in esophageal and gastric carcinogenesis. Among these changes, high frequency of TP53 mutations, loss of heterozygosity (LOH, overexpression of the p53 protein, and consequently loss of p53 function, which would be early events in esophageal and gastric cancers, as well as an important biomarker of the prognosis and treatment response. Furthermore, Single Nucleotide Polymorphisms (SNPs of TP53 have been implicated in the development and prognosis of several cancers, mainly TP53 codon 72 polymorphism whose role has been extensively studied in relation to susceptibility for esophageal and gastric cancer development.

Transcriptome Profiling Reveals the Negative Regulation of Multiple Plant Hormone Signaling Pathways Elicited by Overexpression of C-Repeat Binding Factors

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Aixin Li

2017-09-01

Full Text Available C-repeat binding factors (CBF are a subfamily of AP2 transcription factors that play critical roles in the regulation of plant cold tolerance and growth in low temperature. In the present work, we sought to perform a detailed investigation into global transcriptional regulation of plant hormone signaling associated genes in transgenic plants engineered with CBF genes. RNA samples from Arabidopsis thaliana plants overexpressing two CBF genes, CBF2 and CBF3, were subjected to Illumina HiSeq 2000 RNA sequencing (RNA-Seq. Our results showed that more than half of the hormone associated genes that were differentially expressed in CBF2 or CBF3 transgenic plants were related to auxin signal transduction and metabolism. Most of these alterations in gene expression could lead to repression of auxin signaling. Accordingly, the IAA content was significantly decreased in young tissues of plants overexpressing CBF2 and CBF3 compared with wild type. In addition, genes associated with the biosynthesis of Jasmonate (JA and Salicylic acid (SA, as well as the signal sensing of Brassinolide (BR and SA, were down-regulated, while genes associated with Gibberellin (GA deactivation were up-regulated. In general, overexpression of CBF2 and CBF3 negatively affects multiple plant hormone signaling pathways in Arabidopsis. The transcriptome analysis using CBF2 and CBF3 transgenic plants provides novel and integrated insights into the interaction between CBFs and plant hormones, particularly the modulation of auxin signaling, which may contribute to the improvement of crop yields under abiotic stress via molecular engineering using CBF genes.

Transcriptomic changes reveal gene networks responding to the overexpression of a blueberry DWARF AND DELAYED FLOWERING 1 gene in transgenic blueberry plants.

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Song, Guo-Qing; Gao, Xuan

2017-06-19

Constitutive expression of the CBF/DREB1 for increasing freezing tolerance in woody plants is often associated with other phenotypic changes including dwarf plant and delayed flowering. These phenotypic changes have been observed when Arabidopsis DWARF AND DELAYED FLOWERING 1 (DDF1) was overexpressed in A. thaliana plants. To date, the DDF1 orthologues have not been studied in woody plants. The aim of this study is to investigate transcriptomic responses to the overexpression of blueberry (Vaccinium corymbosum) DDF1 (herein, VcDDF1-OX). The VcDDF1-OX resulted in enhanced freezing tolerance in tetraploid blueberry plants and did not result in significant changes in plant size, chilling requirement, and flowering time. Comparative transcriptome analysis of transgenic 'Legacy-VcDDF1-OX' plants containing an overexpressed VcDDF1 with non-transgenic highbush blueberry 'Legacy' plants revealed the VcDDF1-OX derived differentially expressed (DE) genes and transcripts in the pathways of cold-response, plant flowering, DELLA proteins, and plant phytohormones. The increase in freezing tolerance was associated to the expression of cold-regulated genes (CORs) and the ethylene pathway genes. The unchanged plant size, dormancy and flowering were due to the minimal effect of the VcDDF1-OX on the expression of DELLA proteins, flowering pathway genes, and the other phytohormone genes related to plant growth and development. The DE genes in auxin and cytokinin pathways suggest that the VcDDF1-OX has also altered plant tolerance to drought and high salinity. A DDF1 orthologue in blueberry functioned differently from the DDF1 reported in Arabidopsis. The overexpression of VcDDF1 or its orthologues is a new approach to increase freezing tolerance of deciduous woody plant species with no obvious effect on plant size and plant flowering time.

Nox-2-mediated phenotype loss of hippocampal parvalbumin interneurons might contribute to postoperative cognitive decline in aging mice

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lili qiu

2016-10-01

Full Text Available Postoperative cognitive decline (POCD is a common complication following anesthesia and surgery, especially in elderly patients; however, the precise mechanisms of POCD remain unclear. Here, we investigated whether nicotinamide adenine dinucleotide phosphate (NADPH oxidase mediated-abnormalities in parvalbumin (PV interneurons play an important role in the pathophysiology of POCD. The animal model was established using isoflurane anesthesia and exploratory laparotomy in sixteen-month-old male C57BL/6 mice. For interventional experiments, mice were chronically treated with the NADPH oxidase inhibitor apocynin (APO. Open field and fear conditioning behavioral tests were performed on day 6 and 7 post-surgery, respectively. In a separate experiment, brain tissue was harvested and subjected to biochemical analysis. Primary hippocampal neurons challenged with lipopolysaccharide in vitro were used to investigate the mechanisms underlying the oxidative stress-induced abnormalities in PV interneurons. Our results showed that anesthesia and surgery induced significant hippocampus-dependent memory impairment, which was accompanied by PV interneuron phenotype loss and increased expression of interleukin-1β, markers of oxidative stress, and NADPH oxidase 2 (Nox2 in the hippocampus. In addition, lipopolysaccharide exposure increased Nox2 level and decreased the expression of PV and the number of excitatory synapses onto PV interneurons in the primary hippocampal neurons. Notably, treatment with APO reversed these abnormalities. Our study suggests that Nox2-derived ROS production triggers, at least in part, anesthesia- and surgery-induced hippocampal PV interneuron phenotype loss and consequent cognitive impairment in aging mice.

Synaptic Changes in AMPA Receptor Subunit Expression in Cortical Parvalbumin Interneurons in the Stargazer Model of Absence Epilepsy

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Nadia K. Adotevi

2017-12-01

Full Text Available Feedforward inhibition is essential to prevent run away excitation within the brain. Recent evidence suggests that a loss of feed-forward inhibition in the corticothalamocortical circuitry may underlie some absence seizures. However, it is unclear if this aberration is specifically linked to loss of synaptic excitation onto local fast-spiking parvalbumin-containing (PV+ inhibitory interneurons, which are responsible for mediating feedforward inhibition within cortical networks. We recently reported a global tissue loss of AMPA receptors (AMPARs, and a specific mistrafficking of these AMPARs in PV+ interneurons in the stargazer somatosensory cortex. The current study was aimed at investigating if cellular changes in AMPAR expression were translated into deficits in receptors at specific synapses in the feedforward inhibitory microcircuit. Using western blot immunolabeling on biochemically isolated synaptic fractions, we demonstrate a loss of AMPAR GluA1–4 subunits in the somatosensory cortex of stargazers compared to non-epileptic control mice. Furthermore, using double post-embedding immunogold-cytochemistry, we show a loss of GluA1–4-AMPARs at excitatory synapses onto cortical PV+ interneurons. Altogether, these data indicate a loss of synaptic AMPAR-mediated excitation of cortical PV+ inhibitory neurons. As the cortex is considered the site of initiation of spike wave discharges (SWDs within the corticothalamocortical circuitry, loss of AMPARs at cortical PV+ interneurons likely impairs feed-forward inhibitory output, and contributes to the generation of SWDs and absence seizures in stargazers.

Overexpression of AIB1 in nasopharyngeal carcinomas correlates closely with advanced tumor stage.

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Liu, Meng-Zhong; Xie, Dan; Mai, Shi-Juan; Tong, Zhu-Ting; Shao, Jian-Yong; Fu, Yong-Shui; Xia, Wen-Jie; Kung, Hsian-Fu; Guan, Xin-Yuan; Zeng, Yi-Xin

2008-05-01

AIB1, a candidate oncogene in breast cancer, is commonly amplified and overexpressed in several types of human cancers. In this study, expression and amplification of AIB1 in nasopharyngeal carcinoma (NPC) were studied by immunohistochemical analysis and fluorescence in situ hybridization using tissue microarrays, including 80 specimens of NPC and 20 specimens of nonneoplastic nasopharyngeal mucosa. In this NPC cohort, overexpression and amplification of AIB1 was detected in 36 (51%) of 71 and 3 (7%) of 46 NPCs, respectively. Overexpression of AIB1 was observed more frequently in NPCs in late T stages (T3/T4, 24/35 [69%]) than in earlier stages (T1/T2, 12/36 [33%]; P < .05). In addition, 18 (72%) of 25 NPCs with lymph node metastasis (N1-3) showed overexpression of AIB1; the frequency was significantly higher than that in NPCs without node metastasis (N0, 18/49 [39%]; P < .05). These findings suggest that overexpression of AIB1 in NPCs may be important in the acquisition of an invasive and/or metastatic phenotype.

Overexpression of BIRC6 Is a Predictor of Prognosis for Colorectal Cancer.

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Tingting Hu

Full Text Available Inhibitors of apoptosis proteins (IAPs have been well investigated in human cancers, where they are frequently overexpressed and associated with poor prognosis. Here we explored the role of baculoviral IAP repeat containing 6 (BIRC6, a member of IAPs, in human colorectal cancer (CRC.We used Western blotting and immunohistochemistry to examine BIRC6 expression in 7 CRC cell lines and 126 CRC clinical samples. We determined the biological significance of BIRC6 in CRC cell lines by a lentivirus-mediated silencing method.We reported that BIRC6 was overexpressed in CRC cell lines and clinical CRC tissues. BIRC6 overexpression was correlated with tumor size and invasion depth of CRC. BIRC6 overexpression is associated with worse overall survival (OS (P = 0.001 and shorter disease-free survival (DFS (P = 0.010. BIRC6 knockdown inhibited cell proliferation, arrested cell cycle at S phase, downregulated cyclin A2, B1, D1 and E1 levels, and sensitized CRC cells to chemotherapy in vitro and in vivo.Taken together, these data suggests that BIRC6 overexpression is a predictor of poor prognosis in colorectal cancer and BIRC6 could be a potential target of CRC therapy.

Frequent Nek1 overexpression in human gliomas

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Zhu, Jun [School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China); Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Cai, Yu, E-mail: aihaozuqiu22@163.com [School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China); Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Liu, Pin [Med-X Research Institute, Shanghai Jiao Tong University, Shanghai (China); Zhao, Weiguo [Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China)

2016-08-05

Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients’ poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG, U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. - Highlights: • Nek1 is upregulated in multiple human glioma tissues and cell lines. • Nek1 overexpression correlates with glioma grades and patients’ KPS score. • Nek1 overexpression correlates with patients’ poor overall survival. • siRNA knockdown of Nek1 inhibits glioma cell growth. • siRNA knockdown of Nek1 sensitizes human glioma cells to temozolomide.

Frequent Nek1 overexpression in human gliomas

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Zhu, Jun; Cai, Yu; Liu, Pin; Zhao, Weiguo

2016-01-01

Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients’ poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG, U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. - Highlights: • Nek1 is upregulated in multiple human glioma tissues and cell lines. • Nek1 overexpression correlates with glioma grades and patients’ KPS score. • Nek1 overexpression correlates with patients’ poor overall survival. • siRNA knockdown of Nek1 inhibits glioma cell growth. • siRNA knockdown of Nek1 sensitizes human glioma cells to temozolomide.

Enhanced Arabidopsis pattern-triggered immunity by overexpression of cysteine-rich receptor-like kinases.

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Yeh, Yu-Hung; Chang, Yu-Hsien; Huang, Pin-Yao; Huang, Jing-Bo; Zimmerli, Laurent

2015-01-01

Upon recognition of microbe-associated molecular patterns (MAMPs) such as the bacterial flagellin (or the derived peptide flg22) by pattern-recognition receptors (PRRs) such as the FLAGELLIN SENSING2 (FLS2), plants activate the pattern-triggered immunity (PTI) response. The L-type lectin receptor kinase-VI.2 (LecRK-VI.2) is a positive regulator of Arabidopsis thaliana PTI. Cysteine-rich receptor-like kinases (CRKs) possess two copies of the C-X8-C-X2-C (DUF26) motif in their extracellular domains and are thought to be involved in plant stress resistance, but data about CRK functions are scarce. Here, we show that Arabidopsis overexpressing the LecRK-VI.2-responsive CRK4, CRK6, and CRK36 demonstrated an enhanced PTI response and were resistant to virulent bacteria Pseudomonas syringae pv. tomato DC3000. Notably, the flg22-triggered oxidative burst was primed in CRK4, CRK6, and CRK36 transgenics and up-regulation of the PTI-responsive gene FLG22-INDUCED RECEPTOR-LIKE 1 (FRK1) was potentiated upon flg22 treatment in CRK4 and CRK6 overexpression lines or constitutively increased by CRK36 overexpression. PTI-mediated callose deposition was not affected by overexpression of CRK4 and CRK6, while CRK36 overexpression lines demonstrated constitutive accumulation of callose. In addition, Pst DC3000-mediated stomatal reopening was blocked in CRK4 and CRK36 overexpression lines, while overexpression of CRK6 induced constitutive stomatal closure suggesting a strengthening of stomatal immunity. Finally, bimolecular fluorescence complementation and co-immunoprecipitation analyses in Arabidopsis protoplasts suggested that the plasma membrane localized CRK4, CRK6, and CRK36 associate with the PRR FLS2. Association with FLS2 and the observation that overexpression of CRK4, CRK6, and CRK36 boosts specific PTI outputs and resistance to bacteria suggest a role for these CRKs in Arabidopsis innate immunity.

Muscarinic excitation of parvalbumin-positive interneurons contributes to the severity of pilocarpine-induced seizures

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Yi, Feng; DeCan, Evan; Stoll, Kurt; Marceau, Eric; Deisseroth, Karl; Lawrence, J. Josh

2014-01-01

SUMMARY Objective A common rodent model in epilepsy research employs the muscarinic acetylcholine receptor (mAChR) agonist pilocarpine, yet the mechanisms underlying the induction of pilocarpine-induced seizures (PISs) remain unclear. Global M1 mAChR (M1R) knockout mice are resistant to PISs, implying that M1R activation disrupts excitation/inhibition balance. Parvalbumin-positive (PV) inhibitory neurons express M1 mAChRs, participate in cholinergically-induced oscillations, and can enter a state of depolarization block (DB) during epileptiform activity. Here, we test the hypothesis that pilocarpine activation of M1Rs expressed on PV cells contributes to PISs. Methods CA1 PV cells in PV-CRE mice were visualized with a floxed YFP or hM3Dq-mCherry adeno-associated virus, or by crossing PV-CRE mice with the RosaYFP reporter line. To eliminate M1Rs from PV cells, we generated PV-M1KO mice by crossing PV-CRE and floxed M1 mice. Action potential (AP) frequency was monitored during application of pilocarpine (200 µM). In behavioral experiments, locomotion and seizure symptoms were recorded in WT or PV-M1KO mice during PISs. Results Pilocarpine significantly increased AP frequency in CA1 PV cells into the gamma range. In the continued presence of pilocarpine, a subset (5/7) of PV cells progressed to DB, which was mimicked by hM3Dq activation of Gq-receptor signaling. Pilocarpine-induced depolarization, AP firing at gamma frequency, and progression to DB were prevented in CA1 PV cells of PV-M1KO mice. Finally, compared to WT mice, PV-M1KO mice were associated with reduced severity of PISs. Significance Pilocarpine can directly depolarize PV+ cells via M1R activation but a subset of these cells progress to DB. Our electrophysiological and behavioral results suggest that this mechanism is active during PISs, contributing to a collapse of PV-mediated GABAergic inhibition, dysregulation of excitation/inhibition balance, and increased susceptibility to PISs. PMID:25495999

In1-ghrelin splicing variant is overexpressed in pituitary adenomas and increases their aggressive features

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Ibáñez-Costa, Alejandro; Gahete, Manuel D.; Rivero-Cortés, Esther; Rincón-Fernández, David; Nelson, Richard; Beltrán, Manuel; de la Riva, Andrés; Japón, Miguel A.; Venegas-Moreno, Eva; Gálvez, Ma Ángeles; García-Arnés, Juan A.; Soto-Moreno, Alfonso; Morgan, Jennifer; Tsomaia, Natia; Culler, Michael D.; Dieguez, Carlos; Castaño, Justo P.; Luque, Raúl M.

2015-01-01

Pituitary adenomas comprise a heterogeneous subset of pathologies causing serious comorbidities, which would benefit from identification of novel, common molecular/cellular biomarkers and therapeutic targets. The ghrelin system has been linked to development of certain endocrine-related cancers. Systematic analysis of the presence and functional implications of some components of the ghrelin system, including native ghrelin, receptors and the recently discovered splicing variant In1-ghrelin, in human normal pituitaries (n = 11) and pituitary adenomas (n = 169) revealed that expression pattern of ghrelin system suffers a clear alteration in pituitary adenomasas comparedwith normal pituitary, where In1-ghrelin is markedly overexpressed. Interestingly, in cultured pituitary adenoma cells In1-ghrelin treatment (acylated peptides at 100 nM; 24–72 h) increased GH and ACTH secretion, Ca2+ and ERK1/2 signaling and cell viability, whereas In1-ghrelin silencing (using a specific siRNA; 100 nM) reduced cell viability. These results indicate that an alteration of the ghrelin system, specially its In1-ghrelin variant, could contribute to pathogenesis of different pituitary adenomas types, and suggest that this variant and its related ghrelin system could provide new tools to identify novel, more general diagnostic, prognostic and potential therapeutic targets in pituitary tumors. PMID:25737012

c-MET Overexpression in Colorectal Cancer: A Poor Prognostic Factor for Survival.

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Lee, Su Jin; Lee, Jeeyun; Park, Se Hoon; Park, Joon Oh; Lim, Ho Yeong; Kang, Won Ki; Park, Young Suk; Kim, Seung Tae

2018-03-02

Increased mesenchymal-epithelial transition factor gene (c-MET) expression in several human malignancies is related to increased tumor progression and is a new potential drug target for several types of cancers. In the present study, we investigated the incidence of c-MET overexpression and its prognostic significance in patients with colorectal cancer (CRC). We retrospectively reviewed the data from 255 stage IV CRC patients who had results from a c-MET immunohistochemical test at Samsung Medical Center. We explored the relationships between c-MET overexpression and clinicopathological features and survival. Primary tumor sites were 67 right-sided colon, 98 left-sided colon, and 90 rectum. Forty-two patients (16.7%) had poorly differentiated or mucinous carcinoma. Among the 255 patients, 39 (15.3%) exhibited c-MET overexpression. There was no significant difference in the prevalence of c-MET overexpression according to primary site, histologic differentiation, molecular markers, or metastatic sites. In a comparison of the tumor response to first-line chemotherapy according to the level of c-MET expression, we found no significant difference in either partial response or disease control rate. In the survival analysis, patients with c-MET overexpression had significantly shorter overall survival (39 vs. 27 months; P = .018) and progression-free survival (PFS) during bevacizumab treatment (10 vs. 7 months; P = .024). c-MET overexpression, which was detected in 39 CRC patients (15.3%) irrespective of primary sites or molecular markers, indicated a poor survival prognosis and predicted shorter PFS during bevacizumab treatment in patients with CRC. Further studies are warranted to elucidate the value of c-MET-targeted therapy in CRC patients. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress.

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Ishikawa, Toshiki; Aki, Toshihiko; Yanagisawa, Shuichi; Uchimiya, Hirofumi; Kawai-Yamada, Maki

2015-10-01

BAX INHIBITOR-1 (BI-1) is a cell death suppressor widely conserved in plants and animals. Overexpression of BI-1 enhances tolerance to stress-induced cell death in plant cells, although the molecular mechanism behind this enhancement is unclear. We recently found that Arabidopsis (Arabidopsis thaliana) BI-1 is involved in the metabolism of sphingolipids, such as the synthesis of 2-hydroxy fatty acids, suggesting the involvement of sphingolipids in the cell death regulatory mechanism downstream of BI-1. Here, we show that BI-1 affects cell death-associated components localized in sphingolipid-enriched microdomains of the plasma membrane in rice (Oryza sativa) cells. The amount of 2-hydroxy fatty acid-containing glucosylceramide increased in the detergent-resistant membrane (DRM; a biochemical counterpart of plasma membrane microdomains) fraction obtained from BI-1-overexpressing rice cells. Comparative proteomics analysis showed quantitative changes of DRM proteins in BI-1-overexpressing cells. In particular, the protein abundance of FLOTILLIN HOMOLOG (FLOT) and HYPERSENSITIVE-INDUCED REACTION PROTEIN3 (HIR3) markedly decreased in DRM of BI-1-overexpressing cells. Loss-of-function analysis demonstrated that FLOT and HIR3 are required for cell death by oxidative stress and salicylic acid, suggesting that the decreased levels of these proteins directly contribute to the stress-tolerant phenotypes in BI-1-overexpressing rice cells. These findings provide a novel biological implication of plant membrane microdomains in stress-induced cell death, which is negatively modulated by BI-1 overexpression via decreasing the abundance of a set of key proteins involved in cell death. © 2015 American Society of Plant Biologists. All Rights Reserved.

Overexpression of decorin promoted angiogenesis in diabetic cardiomyopathy via IGF1R-AKT-VEGF signaling.

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Lai, Jinsheng; Chen, Fuqiong; Chen, Jing; Ruan, Guoran; He, Mengying; Chen, Chen; Tang, Jiarong; Wang, Dao Wen

2017-03-14

Microcirculatory dysfunction is believed to play an important role in diabetic cardiomyopathy. The small leucine-rich proteoglycan decorin is generally considered a pro-angiogenic factor. Here, we investigate whether overexpression of decorin ameliorates diabetic cardiomyopathy and its effects on angiogenesis in vivo and in vitro. Diabetes was induced through intraperitoneal injection with streptozotocin combined with a high-fat diet, and decorin was overexpressed via recombinant adeno-associated virus in Wistar rats. Six months later, cardiac function was determined using an echocardiography and cardiac catheter system. The results showed that cardiac function was decreased in diabetic rats and restored by overexpression of decorin. In addition, overexpression of decorin upregulated the expression of VEGF and attenuated the reduction in the cardiac capillary density. In the in vitro study, high glucose induced apoptosis and inhibited the capabilities of tube formation, migration and proliferation, which were all ameliorated by decorin overexpression. Meanwhile, decorin overexpression increased the expression of VEGF and IGF1R, as well as the phosphorylation level of AKT and AP-1. Nonetheless, all of these effects were abolished by pretreatment with the IGF1R antibody or AKT inhibitor. In conclusion, overexpression of decorin ameliorated diabetic cardiomyopathy and promoted angiogenesis through the IGF1R-AKT-VEGF signaling pathway in vivo and in vitro.

Human Epidermal Growth Factor Receptor 2 Overexpression in Micropapillary and Other Variants of Urothelial Carcinoma.

Science.gov (United States)

Behzatoğlu, Kemal; Yörükoğlu, Kutsal; Demir, Hale; Bal, Nebil

2016-06-21

Human epidermal growth factor receptor 2 (HER2) protein overexpression or gene amplification has been shown in urothelial bladder cancer. This could be helpful when using targeted anti-HER2 therapy on these tumors. To evaluate HER2 immunohistochemical expression in conventional urothelial carcinoma (UC), in situ UC, and UC variants primarily in micropapillary urothelial carcinoma (MPUC). The study evaluated 60 MPUC cases; 25 invasive, 20 low-grade noninvasive, and 10 high-grade noninvasive UC cases; 8 in situ UC cases; and 69 UC variant cases. The immunohistochemistry staining was scored according to recommendations of the American Society of Clinical Oncology/College of American Pathologists 2013 HER2 test guideline established for breast cancer and only 3+ staining was considered HER2 overexpression. HER2 overexpression was determined by 3+ staining. 34 of 60 MPUC cases (56%) showed HER2 overexpression (3+ staining). We observed 3+ staining HER2 overexpression in nine of 25 conventional invasive UC cases (36%), four of eight in situ UC cases (50%), and three of six lipid cell variant cases (50%). 3+ staining HER2 overexpression was not seen in eight glandular, six small cell, and five sarcomatoid variant cases. HER2 overexpression was negative in the 20 low-grade noninvasive UC cases but positive in two of the 10 high-grade noninvasive UC cases (20%). We observed HER2 overexpression most commonly in MPUC cases. We also found HER2 overexpression in conventional invasive and in situ UC cases. Pure in situ UC and conventional invasive UC, especially MPUC, could be candidate tumors for treatment with anti-HER2 antibody (trastuzumab therapy). Targeted therapy has a limited place in treatment of bladder cancer. In this study, human epidermal growth factor receptor 2 (HER2) overexpression in bladder carcinomas was evaluated in a large number of cases. Anti-HER2 therapy could be used in bladder cancers, as in breast and gastric cancers. Copyright © 2016 European

Clinical Implications of the Epidermal Growth Factor Receptor overexpression in the High-grade Astrocytomas

International Nuclear Information System (INIS)

Hong, Seong Eon; Kang, Jin Oh; Lee, Hye Kyoung; Yang, Moon Ho; Leem, Won; Cho, Kyung Sam

1996-01-01

To determine the incidence and prognostic effects of EGFR overexpression in the high-grade astrocytomas. With 23 paraffin blocks of the high-garde astrocytomas, expression of EGFR were evaluated by immunohistochemical staining employing polyclonal antibody raised to short cytoplasmic domain of the molecule. Two out of 7 anaplastic astrocytomas and 9 out of 16 glioblastoma multiform patients showed overexpression of EGFR(p=0.44). Three out of 11 patients of age below 55 and 8 out of 12 patients of age over 54 showed EGFR overexpression(p=0.141). Median survival of the EGFR negative anaplastic astrocytoma patient was 37 months. Median survival of the glioblastoma multiform patients were 11 months in EGFR negative group and 7 months in EGFR positive group. But survival difference was not significant(p=0.17). There was a marked trend of increasing overexpression of EGFR in older patients. But survival of the glioblastoma multiform decreased by the overexpression of the EGFR without significant

Overexpression of a novel endogenous NADH kinase in Aspergillus nidulans enhances growth

DEFF Research Database (Denmark)

Panagiotou, Gianni; Grotkjær, Thomas; Hofmann, Gerald

2009-01-01

.7.1.86) has been identified. The enzyme has a predicted molecular weight of 49 kDa. We characterised the role of this NADH kinase by genomic integration of the putative gene AN8837.2 under a strong constitutive promoter. The physiological effects of overexpressed NADH kinase in combination with different...... yield on glucose and the maximum specific growth rate increased from 0.47 g/g and 0.22 h(-1) (wild type) to 0.54 g/g and 0.26 h(-1) (NADH kinase overexpressed), respectively. The results suggest that overexpression of NADH kinase improves the growth efficiency of the cell by increasing the access...

ZEB1 overexpression associated with E-cadherin and microRNA-200 downregulation is characteristic of undifferentiated endometrial carcinoma.

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Romero-Pérez, Laura; López-García, M Ángeles; Díaz-Martín, Juan; Biscuola, Michele; Castilla, M Ángeles; Tafe, Laura J; Garg, Karuna; Oliva, Esther; Matias-Guiu, Xavier; Soslow, Robert A; Palacios, José

2013-11-01

Undifferentiated endometrial carcinomas are very aggressive high-grade endometrial carcinomas that are frequently under-recognized. This study aimed to analyze the molecular alterations underlying the development of these endometrial carcinomas, focusing on those related to dedifferentiation. We assessed a series of 120 tumors: 57 grade 1 and 2 endometrioid endometrial carcinomas, 15 grade 3 endometrioid endometrial carcinomas, 27 endometrial serous carcinomas, and 21 undifferentiated endometrial carcinomas. We found a high frequency of DNA mismatch repair deficiency (38%) and moderate rate of p53 overexpression (∼33%) in undifferentiated carcinomas. In contrast to the characteristic endometrioid phenotype, there was a dramatic downregulation of E-cadherin expression in the undifferentiated subtype. Quantitative methylation studies dismissed CDH1 promoter hypermethylation as the mechanism responsible for this change in gene expression, while immunohistochemistry revealed that the E-cadherin repressor ZEB1 was frequently overexpressed (62%) in undifferentiated endometrial carcinomas. This finding was accompanied by a sharp downregulation in the expression of the miR-200 family of microRNAs, well-known targets of ZEB1. Furthermore, there was enhanced expression of epithelial-to-mesenchymal transition markers in undifferentiated endometrial carcinomas, such as N-cadherin, cytoplasmic p120, and osteonectin. In addition, HMGA2, a regulator of epithelial-to-mesenchymal transition that is expressed in aggressive endometrial tumors, such as endometrial serous carcinomas and carcinosarcomas, was expressed in >20% of undifferentiated carcinomas. These results suggest that ZEB1 overexpression, associated with E-cadherin and miR-200s downregulation, and the expression of mesenchymal markers might enhance the metastatic potential of undifferentiated endometrial carcinomas, leading to a poor prognosis. In addition, our observations suggest that the immnohistochemical analysis

Biologic role of activated leukocyte cell adhesion molecule overexpression in breast cancer cell lines and clinical tumor tissue.

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Hein, Sibyll; Müller, Volkmar; Köhler, Nadine; Wikman, Harriet; Krenkel, Sylke; Streichert, Thomas; Schweizer, Michaela; Riethdorf, Sabine; Assmann, Volker; Ihnen, Maike; Beck, Katrin; Issa, Rana; Jänicke, Fritz; Pantel, Klaus; Milde-Langosch, Karin

2011-09-01

The activated leukocyte cell adhesion molecule (ALCAM) is overexpressed in many mammary tumors, but controversial results about its role and prognostic impact in breast cancer have been reported. Therefore, we evaluated the biologic effects of ALCAM expression in two breast cancer cell lines and a larger cohort of mammary carcinomas. By stable transfections, MCF7 cells with ALCAM overexpression and MDA-MB231 cells with reduced ALCAM levels were generated and analyzed in functional assays and cDNA microarrays. In addition, an immunohistochemical study on 347 patients with breast cancer with long-term follow-up and analysis of disseminated tumor cells (DTCs) was performed. In both cell lines, high ALCAM expression was associated with reduced cell motility. In addition, ALCAM silencing in MDA-MB231 cells resulted in lower invasive potential, whereas high ALCAM expression was associated with increased apoptosis in both cell lines. Among genes which were differentially expressed in clones with altered ALCAM expression, there was an overlap of 15 genes between both cell lines, among them cathepsin D, keratin 7, gelsolin, and ets2 whose deregulation was validated by western blot analysis. In MDA-MB231 cells, we observed a correlation with VEGF expression which was validated by enzyme-linked immuno sorbent assay (ELISA). Our IHC results on primary breast carcinomas showed that ALCAM expression was associated with an estrogen receptor-positive phenotype. In addition, strong ALCAM immunostaining correlated with nodal involvement and the presence of tumor cells in bone marrow. By Kaplan-Meier analysis, strong ALCAM expression in ductal carcinomas correlated with shorter recurrence-free intervals (P=0.048) and overall survival (OAS, P=0.003). Our results indicate that the biologic role of ALCAM in breast cancer is complex, but overexpression might be relevant for outcome in ductal carcinomas.

Overexpressed TP73 induces apoptosis in medulloblastoma

International Nuclear Information System (INIS)

Castellino, Robert C; De Bortoli, Massimiliano; Lin, Linda L; Skapura, Darlene G; Rajan, Jessen A; Adesina, Adekunle M; Perlaky, Laszlo; Irwin, Meredith S; Kim, John YH

2007-01-01

Medulloblastoma is the most common malignant brain tumor of childhood. Children who relapse usually die of their disease, which reflects resistance to radiation and/or chemotherapy. Improvements in outcome require a better understanding of the molecular basis of medulloblastoma growth and treatment response. TP73 is a member of the TP53 tumor suppressor gene family that has been found to be overexpressed in a variety of tumors and mediates apoptotic responses to genotoxic stress. In this study, we assessed expression of TP73 RNA species in patient tumor specimens and in medulloblastoma cell lines, and manipulated expression of full-length TAp73 and amino-terminal truncated ΔNp73 to assess their effects on growth. We analyzed medulloblastoma samples from thirty-four pediatric patients and the established medulloblastoma cell lines, Daoy and D283MED, for expression of TP73 RNA including the full-length transcript and the 5'-terminal variants that encode the ΔNp73 isoform, as well as TP53 RNA using quantitative real time-RTPCR. Protein expression of TAp73 and ΔNp73 was quantitated with immunoblotting methods. Clinical outcome was analyzed based on TP73 RNA and p53 protein expression. To determine effects of overexpression or knock-down of TAp73 and ΔNp73 on cell cycle and apoptosis, we analyzed transiently transfected medulloblastoma cell lines with flow cytometric and TUNEL methods. Patient medulloblastoma samples and cell lines expressed full-length and 5'-terminal variant TP73 RNA species in 100-fold excess compared to non-neoplastic brain controls. Western immunoblot analysis confirmed their elevated levels of TAp73 and amino-terminal truncated ΔNp73 proteins. Kaplan-Meier analysis revealed trends toward favorable overall and progression-free survival of patients whose tumors display TAp73 RNA overexpression. Overexpression of TAp73 or ΔNp73 induced apoptosis under basal growth conditions in vitro and sensitized them to cell death in response to

Overexpressed TP73 induces apoptosis in medulloblastoma

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Perlaky Laszlo

2007-07-01

Full Text Available Abstract Background Medulloblastoma is the most common malignant brain tumor of childhood. Children who relapse usually die of their disease, which reflects resistance to radiation and/or chemotherapy. Improvements in outcome require a better understanding of the molecular basis of medulloblastoma growth and treatment response. TP73 is a member of the TP53 tumor suppressor gene family that has been found to be overexpressed in a variety of tumors and mediates apoptotic responses to genotoxic stress. In this study, we assessed expression of TP73 RNA species in patient tumor specimens and in medulloblastoma cell lines, and manipulated expression of full-length TAp73 and amino-terminal truncated ΔNp73 to assess their effects on growth. Methods We analyzed medulloblastoma samples from thirty-four pediatric patients and the established medulloblastoma cell lines, Daoy and D283MED, for expression of TP73 RNA including the full-length transcript and the 5'-terminal variants that encode the ΔNp73 isoform, as well as TP53 RNA using quantitative real time-RTPCR. Protein expression of TAp73 and ΔNp73 was quantitated with immunoblotting methods. Clinical outcome was analyzed based on TP73 RNA and p53 protein expression. To determine effects of overexpression or knock-down of TAp73 and ΔNp73 on cell cycle and apoptosis, we analyzed transiently transfected medulloblastoma cell lines with flow cytometric and TUNEL methods. Results Patient medulloblastoma samples and cell lines expressed full-length and 5'-terminal variant TP73 RNA species in 100-fold excess compared to non-neoplastic brain controls. Western immunoblot analysis confirmed their elevated levels of TAp73 and amino-terminal truncated ΔNp73 proteins. Kaplan-Meier analysis revealed trends toward favorable overall and progression-free survival of patients whose tumors display TAp73 RNA overexpression. Overexpression of TAp73 or ΔNp73 induced apoptosis under basal growth conditions in vitro and

Effects of GGCX overexpression on anterior cruciate ligament transection-induced osteoarthritis in rabbits.

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Fu, Xiaoling; Qiu, Ruiyun; Tang, Chunfang; Wang, Xiaomei; Cheng, Xigao; Yin, Ming

2018-03-01

Effective therapeutic methods for osteoarthritis (OA) are lacking. γ‑glutamyl carboxylase (GGCX) is a key enzyme that regulates carboxylation of cartilage matrix Gla protein (MGP). Whether GGCX overexpression protects against OA remains unknown. The aim of the present study was to explore the effects of GGCX overexpression on anterior cruciate ligament transection (ACLT)‑induced OA and its mechanisms in Japanese white rabbits. ACLT surgery was used to establish an OA model in rabbits. A total of 48 rabbits were randomly divided into 4 groups: Sham, OA model + GGCX overexpression plasmid, OA model + saline and OA model + empty vector. The expression of uncarboxylated MGP (ucMGP), carboxylated MGP (cMGP), matrix metalloproteinase (MMP)‑13, collagen type X, collagen type II, tumor necrosis factor (TNF)‑α and interleukin (IL)‑1β were detected by ELISA, immunohistochemistry, reverse transcription‑quantitative polymerase chain reaction and western blotting. Morphological changes to tibial cartilage were assessed by Giemsa and safranin O‑fast green staining, respectively. Compared with the Sham control, GGCX expression was significantly decreased in the OA Model group. GGCX expression was increased by injection of a lentivirus‑carried overexpression plasmid that encoded GGCX. GGCX overexpression ameliorated ATLC‑induced damage in articular cartilage. OA Model rabbits exhibited significantly decreased expression levels of cMGP and collagen type II, and increased expression of ucMGP, collagen type X, MMP‑13, IL‑1β and TNF‑α. Notably, these expression levels were reversed by GGCX overexpression in OA Model rabbits. Results from the present study indicated that GGCX expression was decreased in OA Model rabbits, whereas overexpression of GGCX was able to promote carboxylation of MGP, reduce inflammation, decrease MMP‑13 expression and regulate collagen expression. The results also indicated that GGCX may serve as a therapeutic target for OA.

Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

International Nuclear Information System (INIS)

Lu, Li; Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin; Song, Wen-Hui; Yan, Ba-Yi; Yang, Gui-Jiao; Li, Ang; Yang, Wu-Lin

2014-01-01

Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5

Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

Energy Technology Data Exchange (ETDEWEB)

Lu, Li, E-mail: luli7300@126.com [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Wen-Hui [Department of Orthopaedics, The Second Affiliated Hospital of Shanxi Medical University, Taiyuan 030001 (China); Yan, Ba-Yi; Yang, Gui-Jiao [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Li, Ang [Department of Medicine, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Department of Anatomy, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Yang, Wu-Lin, E-mail: wulinyoung@163.com [School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium (SBIC), Agency for Science, Technology and Research - A*STAR (Singapore)

2014-01-24

Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.

Attenuation of teratoma formation by p27 overexpression in induced pluripotent stem cells.

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Matsu-ura, Toru; Sasaki, Hiroshi; Okada, Motoi; Mikoshiba, Katsuhiko; Ashraf, Muhammad

2016-02-15

Pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells, have a great potential for regenerative medicine. Induced pluripotent stem cells, in particular, are suitable for replacement of tissue by autologous transplantation. However, tumorigenicity is a major risk in clinical application of both embryonic stem cells and induced pluripotent stem cells. This study explores the possibility of manipulating the cell cycle for inhibition of tumorigenicity. We genetically modified mouse induced pluripotent stem cells (miPSCs) to overexpress p27 tumor suppressor and examined their proliferation rate, gene expression, cardiac differentiation, tumorigenicity, and therapeutic potential in a mouse model of coronary artery ligation. Overexpression of p27 inhibited cell division of miPSCs, and that inhibition was dependent on the expression level of p27. p27 overexpressing miPSCs had pluripotency characteristics but lost stemness earlier than normal miPSCs during embryoid body and teratoma formation. These cellular characteristics led to none or smaller teratoma when the cells were injected into nude mice. Transplantation of both miPSCs and p27 overexpressing miPSCs into the infarcted mouse heart reduced the infarction size and improved left ventricular function. The overexpression of p27 attenuated tumorigenicity by reducing proliferation and earlier loss of stemness of miPSCs. The overexpression of p27 did not affect pluripotency and differentiation characteristics of miPSC. Therefore, regulation of the proliferation rate of miPSCs offers great therapeutic potential for repair of the injured myocardium.

Overexpression of allene oxide cyclase improves the biosynthesis of artemisinin in Artemisia annua L.

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Xu Lu

Full Text Available Jasmonates (JAs are important signaling molecules in plants and play crucial roles in stress responses, secondary metabolites' regulation, plant growth and development. In this study, the promoter of AaAOC, which was the key gene of jasmonate biosynthetic pathway, had been cloned. GUS staining showed that AaAOC was expressed ubiquitiously in A. annua. AaAOC gene was overexpressed under control of 35S promoter. RT-Q-PCR showed that the expression levels of AaAOC were increased from 1.6- to 5.2-fold in AaAOC-overexpression transgenic A. annua. The results of GC-MS showed that the content of endogenous jasmonic acid (JA was 2- to 4.7-fold of the control level in AaAOC-overexpression plants. HPLC showed that the contents of artemisinin, dihydroartemisinic acid and artemisinic acid were increased significantly in AaAOC-overexpression plants. RT-Q-PCR showed that the expression levels of FPS (farnesyl diphosphate synthase, CYP71AV1 (cytochrome P450 dependent hydroxylase and DBR2 (double bond reductase 2 were increased significantly in AaAOC-overexpression plants. All data demonstrated that increased endogenous JA could significantly promote the biosynthesis of artemisinin in AaAOC-overexpression transgenic A. annua.

Effect of Rad 51 overexpression on chromosomal stability and radiation sensitivity in tumour cells

International Nuclear Information System (INIS)

Jend, C.; Stuerzbecher, H.W.; Dikomey, E.; Borgmann, K.

2004-01-01

The present study was dedicated to examining the effects of Rad51 overexpression on genomic instability, expressed in terms of chromosomal aberrations in G1 and G2 phases following X-ray irradiation. For this purpose an osteosarcoma cell line (Ui-OS) which shows inducing Rad51 overexpression (UiRad5-2) after stable transfection was compared with an isogenetic line (UiLacZ) which overexpresses beta-galactosidase instead of Rad51 [de

Transcription factors and molecular epigenetic marks underlying EpCAM overexpression in ovarian cancer

NARCIS (Netherlands)

van der Gun, B. T. F.; de Groote, M. L.; Kazemier, H. G.; Arendzen, A. J.; Terpstra, P.; Ruiters, M. H. J.; McLaughlin, P. M. J.; Rots, M. G.

2011-01-01

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is overexpressed on carcinomas, and its downregulation inhibits the oncogenic potential of multiple tumour types. Here, we investigated underlying mechanisms of epcam overexpression in ovarian carcinoma. METHODS: Expression of EpCAM and DNA

MDMA-induced loss of parvalbumin interneurons within the dentate gyrus is mediated by 5HT2A and NMDA receptors.

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Collins, Stuart A; Gudelsky, Gary A; Yamamoto, Bryan K

2015-08-15

MDMA is a widely abused psychostimulant which causes a rapid and robust release of the monoaminergic neurotransmitters dopamine and serotonin. Recently, it was shown that MDMA increases extracellular glutamate concentrations in the dorsal hippocampus, which is dependent on serotonin release and 5HT2A/2C receptor activation. The increased extracellular glutamate concentration coincides with a loss of parvalbumin-immunoreactive (PV-IR) interneurons of the dentate gyrus region. Given the known susceptibility of PV interneurons to excitotoxicity, we examined whether MDMA-induced increases in extracellular glutamate in the dentate gyrus are necessary for the loss of PV cells in rats. Extracellular glutamate concentrations increased in the dentate gyrus during systemic and local administration of MDMA. Administration of the NMDA receptor antagonist, MK-801, during systemic injections of MDMA, prevented the loss of PV-IR interneurons seen 10 days after MDMA exposure. Local administration of MDL100907, a selective 5HT2A receptor antagonist, prevented the increases in glutamate caused by reverse dialysis of MDMA directly into the dentate gyrus and prevented the reduction of PV-IR. These findings provide evidence that MDMA causes decreases in PV within the dentate gyrus through a 5HT2A receptor-mediated increase in glutamate and subsequent NMDA receptor activation. Copyright © 2015 Elsevier B.V. All rights reserved.

Lack of Cdkl5 disrupts the organization of excitatory and inhibitory synapses and parvalbumin interneurons in the primary visual cortex

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Riccardo Pizzo

2016-11-01

Full Text Available CDKL5 (cyclin-dependent kinase-like 5 mutations are found in severe neurodevelopmental disorders, including the Hanefeld variant of Rett syndrome (CDKL5 disorder. CDKL5 loss-of-function murine models recapitulate pathological signs of the human disease, such as visual attention deficits and reduced visual acuity. Here we investigated the cellular and synaptic substrates of visual defects by studying the organization of the primary visual cortex (V1 of Cdkl5-/y mice. We found a severe reduction of c-fos expression in V1 of Cdkl5-/y mutants, suggesting circuit hypoactivity. Glutamatergic presynaptic structures were increased, but postsynaptic PSD-95 and Homer were significantly downregulated in CDKL5 mutants. Interneurons expressing parvalbumin, but not other types of interneuron, had a higher density in mutant V1, and were hyperconnected with pyramidal neurons. Finally, the developmental trajectory of pavalbumin-containing cells was also affected in Cdkl5-/y mice, as revealed by fainter appearance perineuronal nets at the closure of the critical period. The present data reveal an overall disruption of V1 cellular and synaptic organization that may cause a shift in the excitation/inhibition balance likely to underlie the visual deficits characteristic of CDKL5 disorder. Moreover, ablation of CDKL5 is likely to tamper with the mechanisms underlying experience-dependent refinement of cortical circuits during the critical period of development.

Lack of Cdkl5 Disrupts the Organization of Excitatory and Inhibitory Synapses and Parvalbumin Interneurons in the Primary Visual Cortex.

Science.gov (United States)

Pizzo, Riccardo; Gurgone, Antonia; Castroflorio, Enrico; Amendola, Elena; Gross, Cornelius; Sassoè-Pognetto, Marco; Giustetto, Maurizio

2016-01-01

Cyclin-dependent kinase-like 5 (CDKL5) mutations are found in severe neurodevelopmental disorders, including the Hanefeld variant of Rett syndrome (RTT; CDKL5 disorder). CDKL5 loss-of-function murine models recapitulate pathological signs of the human disease, such as visual attention deficits and reduced visual acuity. Here we investigated the cellular and synaptic substrates of visual defects by studying the organization of the primary visual cortex (V1) of Cdkl5 -/y mice. We found a severe reduction of c-Fos expression in V1 of Cdkl5 -/y mutants, suggesting circuit hypoactivity. Glutamatergic presynaptic structures were increased, but postsynaptic PSD-95 and Homer were significantly downregulated in CDKL5 mutants. Interneurons expressing parvalbumin, but not other types of interneuron, had a higher density in mutant V1, and were hyperconnected with pyramidal neurons. Finally, the developmental trajectory of pavalbumin-containing cells was also affected in Cdkl5 -/y mice, as revealed by fainter appearance perineuronal nets at the closure of the critical period (CP). The present data reveal an overall disruption of V1 cellular and synaptic organization that may cause a shift in the excitation/inhibition balance likely to underlie the visual deficits characteristic of CDKL5 disorder. Moreover, ablation of CDKL5 is likely to tamper with the mechanisms underlying experience-dependent refinement of cortical circuits during the CP of development.

Overexpression of cellular glutathione peroxidase rescues homocyst(e)ine-induced endothelial dysfunction

Science.gov (United States)

Weiss, Norbert; Zhang, Ying-Yi; Heydrick, Stanley; Bierl, Charlene; Loscalzo, Joseph

2001-01-01

Homocyst(e)ine (Hcy) inhibits the expression of the antioxidant enzyme cellular glutathione peroxidase (GPx-1) in vitro and in vivo, which can lead to an increase in reactive oxygen species that inactivate NO and promote endothelial dysfunction. In this study, we tested the hypothesis that overexpression of GPx-1 can restore the normal endothelial phenotype in hyperhomocyst(e)inemic states. Heterozygous cystathionine β-synthase-deficient (CBS(−/+)) mice and their wild-type littermates (CBS(+/+)) were crossbred with mice that overexpress GPx-1 [GPx-1(tg+) mice]. GPx-1 activity was 28% lower in CBS(−/+)/GPx-1(tg−) compared with CBS(+/+)/GPx-1(tg−) mice (P < 0.05), and CBS(−/+) and CBS(+/+) mice overexpressing GPx-1 had 1.5-fold higher GPx-1 activity compared with GPx-1 nontransgenic mice (P < 0.05). Mesenteric arterioles of CBS(−/+)/GPx-1(tg−) mice showed vasoconstriction to superfusion with β-methacholine and bradykinin (P < 0.001 vs. all other groups), whereas nonhyperhomocyst(e)inemic mice [CBS(+/+)/GPx-1(tg−) and CBS(+/+)/GPx-1(tg+) mice] demonstrated dose-dependent vasodilation in response to both agonists. Overexpression of GPx-1 in hyperhomocyst(e)inemic mice restored the normal endothelium-dependent vasodilator response. Bovine aortic endothelial cells (BAEC) were transiently transfected with GPx-1 and incubated with dl-homocysteine (HcyH) or l-cysteine. HcyH incubation decreased GPx-1 activity in sham-transfected BAEC (P < 0.005) but not in GPx-1-transfected cells. Nitric oxide release from BAEC was significantly decreased by HcyH but not cysteine, and GPx-1 overexpression attenuated this decrease. These findings demonstrate that overexpression of GPx-1 can compensate for the adverse effects of Hcy on endothelial function and suggest that the adverse vascular effects of Hcy are at least partly mediated by oxidative inactivation of NO. PMID:11606774

Voronoi-based spatial analysis reveals selective interneuron changes in the cortex of FALS mice.

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Minciacchi, Diego; Kassa, Roman M; Del Tongo, Claudia; Mariotti, Raffaella; Bentivoglio, Marina

2009-01-01

The neurodegenerative disease amyotrophic lateral sclerosis affects lower motoneurons and corticospinal cells. Mice expressing human mutant superoxide dismutase (SOD)1 provide widely investigated models of the familial form of disease, but information on cortical changes in these mice is still limited. We here analyzed the spatial organization of interneurons characterized by parvalbumin immunoreactivity in the motor, somatosensory, and visual cortical areas of SOD1(G93A) mice. Cell number and sociological spatial behavior were assessed by digital charts of cell location in cortical samples, cell counts, and generation of two-dimensional Voronoi diagrams. In end-stage SOD1-mutant mice, an increase of parvalbumin-containing cortical interneurons was found in the motor and somatosensory areas (about 35% and 20%, respectively) with respect to wild-type littermates. Changes in cell spatial distribution, as documented by Voronoi-derived coefficients of variation, indicated increased tendency of parvalbumin cells to aggregate into clusters in the same areas of the SOD1-mutant cortex. Counts and coefficients of variation of parvalbumin cells in the visual cortex gave instead similar results in SOD1-mutant and wild-type mice. Analyses of motor and somatosensory areas in presymptomatic SOD1-mutant mice provided findings very similar to those obtained at end-stage, indicating early changes of interneurons in these cortical areas during the pathology. Altogether the data reveal in the SOD1-mutant mouse cortex an altered architectonic pattern of interneurons, which selectively affects areas involved in motor control. The findings, which can be interpreted as pathogenic factors or early disease-related adaptations, point to changes in the cortical regulation and modulation of the motor circuit during motoneuron disease.

Effects of industrial processing on the immunogenicity of commonly ingested fish species.

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Sletten, Gaynour; Van Do, Thien; Lindvik, Helene; Egaas, Eliann; Florvaag, Erik

2010-01-01

Food-processing techniques may induce changes in fish protein immunogenicity. Allergens from >100 fish species have been identified, but little is known on the effects of processing on fish protein immunogenicity. IgE binding of sera of patients allergic to fresh and processed (smoked, salted/sugar-cured, canned, lye-treated and fermented) cod, haddock, salmon, trout, tuna, mackerel and herring and of hydrolysates based on salmon and whiting was investigated using immunoblot and inhibition ELISA. Parvalbumin oligomers were identified using monoclonal and polyclonal antibodies. IgE binding was seen in most sera at 12-14 kDa (parvalbumin), and at 17-60 kDa for all fish except tuna. Changes in IgE binding appeared to reflect altered parvalbumin monomers and oligomers. Smoked haddock, salmon and mackerel had increased IgE binding and novel bands at 30 kDa. Chemically processed cod, salmon, trout and pickled herring had reduced or abolished IgE binding. The serum of 1 subject, however, had increased IgE binding to these products and also inhibition of binding by both fish hydrolysates to their constituent fish species. Process-induced changes in fish protein immunogenicity were more dependent on process rather than species, although individual responses varied. Changes in the allergenicity of a product may depend on the net effect of processing on parvalbumin oligomerization patterns, which may also vary in different species. Chemical processes generally caused loss in IgE-binding activity, though sensitization may occur to modified or degraded rather than intact peptides as shown by increased binding by chemically processed fish and hydrolysates in 1 subject. The clinical significance of these findings remains to be established.

Adenosine Inhibits the Excitatory Synaptic Inputs to Basal Forebrain Cholinergic, GABAergic and Parvalbumin Neurons in mice

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Chun eYang

2013-06-01

Full Text Available Coffee and tea contain the stimulants caffeine and theophylline. These compounds act as antagonists of adenosine receptors. Adenosine promotes sleep and its extracellular concentration rises in association with prolonged wakefulness, particularly in the basal forebrain (BF region involved in activating the cerebral cortex. However, the effect of adenosine on identified BF neurons, especially non-cholinergic neurons, is incompletely understood. Here we used whole-cell patch-clamp recordings in mouse brain slices prepared from two validated transgenic mouse lines with fluorescent proteins expressed in GABAergic or parvalbumin (PV neurons to determine the effect of adenosine. Whole-cell recordings were made BF cholinergic neurons and from BF GABAergic & PV neurons with the size (>20 µm and intrinsic membrane properties (prominent H-currents corresponding to cortically projecting neurons. A brief (2 min bath application of adenosine (100 μM decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents in all groups of BF cholinergic, GABAergic and PV neurons we recorded. In addition, adenosine decreased the frequency of miniature EPSCs in BF cholinergic neurons. Adenosine had no effect on the frequency of spontaneous inhibitory postsynaptic currents in cholinergic neurons or GABAergic neurons with large H-currents but reduced them in a group of GABAergic neurons with smaller H-currents. All effects of adenosine were blocked by a selective, adenosine A1 receptor antagonist, cyclopentyltheophylline (CPT, 1 μM. Adenosine had no postsynaptic effects. Taken together, our work suggests that adenosine promotes sleep by an A1-receptor mediated inhibition of glutamatergic inputs to cortically-projecting cholinergic and GABA/PV neurons. Conversely, caffeine and theophylline promote attentive wakefulness by inhibiting these A1 receptors in BF thereby promoting the high-frequency oscillations in the cortex required for

Altered microRNA signatures in sputum of patients with active pulmonary tuberculosis.

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Zhengjun Yi

Full Text Available Role of microRNA (miRNA has been highlighted in pathogen-host interactions recently. At present, their role in active pulmonary tuberculosis is unknown. The aim of the study was to delineate miRNA expression in sputum supernatant of patients with active pulmonary tuberculosis. Expression of miRNAs was evaluated by microarray analysis and differentially expressed miRNAs were validated by RT-qPCR. Secreted cytokines TNF-α and IL-6 were measured by ELISA. We found that 95 miRNAs were differentially expressed between tuberculosis group and controls. More miRNAs (52 out of 95 miRNAs were underexpressed than overexpressed during tuberculosis infection. Overexpression of miR-3179, miR-147 and underexpression of miR-19b-2* in TB group compared with controls were confirmed in the validation cohort. TNF-α and IL-6 levels were not significantly altered between TB group and controls. For the first time, differential expression of miRNAs in sputum was found in active pulmonary tuberculosis. The study provides rationale for identifying the role of miRNAs in the pathogenesis of pulmonary tuberculosis and indicates potential for miRNA-based therapeutic strategies.

Altered microRNA signatures in sputum of patients with active pulmonary tuberculosis.

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Yi, Zhengjun; Fu, Yurong; Ji, Rui; Li, Ruifang; Guan, Zhiyu

2012-01-01

Role of microRNA (miRNA) has been highlighted in pathogen-host interactions recently. At present, their role in active pulmonary tuberculosis is unknown. The aim of the study was to delineate miRNA expression in sputum supernatant of patients with active pulmonary tuberculosis. Expression of miRNAs was evaluated by microarray analysis and differentially expressed miRNAs were validated by RT-qPCR. Secreted cytokines TNF-α and IL-6 were measured by ELISA. We found that 95 miRNAs were differentially expressed between tuberculosis group and controls. More miRNAs (52 out of 95 miRNAs) were underexpressed than overexpressed during tuberculosis infection. Overexpression of miR-3179, miR-147 and underexpression of miR-19b-2* in TB group compared with controls were confirmed in the validation cohort. TNF-α and IL-6 levels were not significantly altered between TB group and controls. For the first time, differential expression of miRNAs in sputum was found in active pulmonary tuberculosis. The study provides rationale for identifying the role of miRNAs in the pathogenesis of pulmonary tuberculosis and indicates potential for miRNA-based therapeutic strategies.

Bcl-2 overexpression prevents 99mTc-MIBI uptake in breast cancer cell lines

International Nuclear Information System (INIS)

Aloj, Luigi; Zannetti, Antonella; Caraco, Corradina; Del Vecchio, Silvana; Salvatore, Marco

2004-01-01

We have previously shown a correlation between the absence of technetium-99m methoxyisobutylisonitrile ( 99m Tc-MIBI) uptake and overexpression of the anti-apoptotic protein Bcl-2 in human breast carcinoma. To establish a direct cause-effect relationship between Bcl-2 overexpression and reduced 99m Tc-MIBI uptake, MCF-7 and T47D breast cancer cell lines were stably transfected with the human Bcl-2 gene to increase intracellular protein levels and tested for 99m Tc-MIBI uptake. All clones overexpressing Bcl-2 showed a dramatic reduction of 99m Tc-MIBI uptake as compared with mock transfected control cells. Tracer uptake was promptly and partially restored by induction of apoptosis with staurosporine treatment. After 4.5 h of staurosporine treatment, a tenfold increase in 99m Tc-MIBI uptake was observed in treated as compared with untreated Bcl-2 overexpressing cells. Our findings provide a rational basis for the development of an in vivo test to detect Bcl-2 overexpression in human tumours. (orig.)

Ectoderm-targeted overexpression of the glucocorticoid receptor induces hypohidrotic ectodermal dysplasia.

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Cascallana, Jose Luis; Bravo, Ana; Donet, Eva; Leis, Hugo; Lara, Maria Fernanda; Paramio, Jesús M; Jorcano, José L; Pérez, Paloma

2005-06-01

Hypohidrotic ectodermal dysplasia is a human syndrome defined by maldevelopment of one or more ectodermal-derived tissues, including the epidermis and cutaneous appendices, teeth, and exocrine glands. The molecular bases of this pathology converge in a dysfunction of the transcription factor nuclear factor of the kappa-enhancer in B cells (NF-kappaB), which is essential to epithelial homeostasis and development. A number of mouse models bearing disruptions in NF-kappaB signaling have been reported to manifest defects in ectodermal derivatives. In ectoderm-targeted transgenic mice overexpressing the glucocorticoid receptor (GR) [keratin 5 (K5)-GR mice], the NF-kappaB activity is greatly decreased due to functional antagonism between GR and NF-kappaB. Here, we report that K5-GR mice exhibit multiple epithelial defects in hair follicle, tooth, and palate development. Additionally, these mice lack Meibomian glands and display underdeveloped sweat and preputial glands. These phenotypic features appear to be mediated specifically by ligand-activated GR because the synthetic analog dexamethasone induced similar defects in epithelial morphogenesis, including odontogenesis, in wild-type mice. We have focused on tooth development in K5-GR mice and found that an inhibitor of steroid synthesis partially reversed the abnormal phenotype. Immunostaining revealed reduced expression of the inhibitor of kappaB kinase subunits, IKKalpha and IKKgamma, and diminished p65 protein levels in K5-GR embryonic tooth, resulting in a significantly reduced kappaB-binding activity. Remarkably, altered NF-kappaB activity elicited by GR overexpression correlated with a dramatic decrease in the protein levels of DeltaNp63 in tooth epithelia without affecting Akt, BMP4, or Foxo3a. Given that many of the 170 clinically distinct ectodermal dysplasia syndromes still remain without cognate genes, deciphering the molecular mechanisms of this mouse model with epithelial NF-kappaB and p63 dysfunction may

Characterization of MCF mammary epithelial cells overexpressing the Arylhydrocarbon receptor (AhR)

International Nuclear Information System (INIS)

Wong, Patrick S; Li, Wen; Vogel, Christoph F; Matsumura, Fumio

2009-01-01

Recent reports indicate the existence of breast cancer cells expressing very high levels of the Arylhydrocarbon receptor (AhR), a ubiquitous intracellular receptor best known for mediating toxic action of dioxin and related pollutants. Positive correlation between the degree of AhR overexpression and states of increasing transformation of mammary epithelial cells appears to occur in the absence of any exogenous AhR ligands. These observations have raised many questions such as why and how AhR is overexpressed in breast cancer and its physiological roles in the progression to advanced carcinogenic transformation. To address those questions, we hypothesized that AhR overexpression occurs in cells experiencing deficiencies in normally required estrogen receptor (ER) signaling, and the basic role of AhR in such cases is to guide the affected cells to develop orchestrated cellular changes aimed at substituting the normal functions of ER. At the same time, the AhR serves as the mediator of the cell survival program in the absence of ER signaling. We subjected two lines of Michigan Cancer Foundation (MCF) mammary epithelial cells to 3 different types ER interacting agents for a number of passages and followed the changes in the expression of AhR mRNA. The resulting sublines were analyzed for phenotypical changes and unique molecular characteristics. MCF10AT1 cells continuously exposed to 17-beta-estradiol (E2) developed sub-lines that show AhR overexpression with the characteristic phenotype of increased proliferation, and distinct resistance to apoptosis. When these chemically selected cell lines were treated with a specific AhR antagonist, 3-methoxy-4-nitroflavone (MNF), both of the above abnormal cellular characteristics disappeared, indicating the pivotal role of AhR in expressing those cellular phenotypes. The most prominent molecular characteristics of these AhR overexpressing MCF cells were found to be overexpression of ErbB2 and COX-2. Furthermore, we could

AICD Overexpression in Neuro 2A Cells Regulates Expression of PTCH1 and TRPC5

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Mithu Raychaudhuri

2011-01-01

Full Text Available Amyloid precursor protein (APP, implicated in Alzheimer's disease, is a transmembrane protein of undetermined function. APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD in the cytoplasm. In vitro and in vivo studies have implicated the role of AICD in cell signaling and transcriptional regulation of Gsk3β, KAI1, BACE1, EGFR, and other proteins. In this study, by overexpressing AICD in mouse neuroblastoma cell lines, we have demonstrated the alteration in the expressions of two proteins, patched homolog 1 (PTCH1, a receptor for sonic hedgehog signaling, and transient receptor potential cation channel subfamily C member 5 (TRPC5, a component of receptor-activated nonselective calcium permeant cation channel. Our results indicate the possibility of regulation by AICD in developmental processes as well as in the maintenance of calcium homeostasis at the transcription level.

Calretinin and parvalbumin immunoreactive interneurons in the retrosplenial cortex of the rat brain: Qualitative and quantitative analyses.

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Salaj, Martin; Druga, Rastislav; Cerman, Jiří; Kubová, Hana; Barinka, Filip

2015-11-19

The retrosplenial cortex (RSC) is a mesocortical region broadly involved with memory and navigation. It shares many characteristics with the perirhinal cortex (PRC), both of which appear to be significantly involved in the spreading of epileptic activity. We hypothesized that RSC possesses an interneuronal composition similar to that of PRC. To prove the hypothesis we studied the general pattern of calretinin (CR) and parvalbumin (PV) immunoreactivity in the RSC of the rat brain, its optical density as well as the morphological features and density of CR- and PV-immunoreactive (CR+ and PV+) interneurons. We also analyzed the overall neuronal density on Nissl-stained sections in RSC. Finally, we compared our results with our earlier analysis of PRC (Barinka et al., 2012). Compared to PRC, RSC was observed to have a higher intensity of PV staining and lower intensity of CR staining of neuropil. Vertically-oriented bipolar neurons were the most common morphological type among CR+ neurons. The staining pattern did not allow for a similarly detailed analysis of somatodendritic morphology of PV+ neurons. RSC possessed lower absolute (i.e., neurons/mm(3)) and relative (i.e., percentage of the overall neuronal population) densities of CR+ neurons and similar absolute and lower relative densities of PV+ neurons relative to PRC. CR: PV neuronal ratio in RSC (1:2 in area 29 and 1:2.2 in area 30) differed from PRC (1:1.2 in area 35 and 1:1.7 in area 36). In conclusion, RSC, although similar in many aspects to PRC, differs strikingly in the interneuronal composition relative to PRC. Copyright © 2015 Elsevier B.V. All rights reserved.

Transgenic overexpression of BAFF regulates the expression of ...

Indian Academy of Sciences (India)

To investigate whether transgenic overexpression of the zebrafish BAFF leads to ... and BAFF proteins were expressed separately and confirmed in HeLa cells. ... body homogenate of zebrafish and demonstrated a significant increase in ...

Overexpression of NIMA-related kinase 2 is associated with poor prognoses in malignant glioma.

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Liu, Huajie; Liu, Bin; Hou, Xianzeng; Pang, Bo; Guo, Pengbo; Jiang, Wanli; Ding, Qian; Zhang, Rui; Xin, Tao; Guo, Hua; Xu, Shangchen; Pang, Qi

2017-05-01

Eleated expression of NIMA-related kinase 2 (NEK2) was frequently observed in a variety of malignant cancers, and it appears to be involved in the initiation, maintenance, progression, metastasis of cancer and is positively associated with poor prognosis. We sought to investigate NEK2 expression and its predictive roles in malignant gliomas, and study the correlation of NEK2 protein expression with proliferation, clinical parameters, overall survival and some other parameters. We investigate NEK2 protein expression in 99 samples of malignant gliomas, including 35 WHO grade II, 22 grade III, and 42 grade IV gliomas, by immunohistochemistry and western blot (n = 50). We then made correlative analysis of protein overexpression using the Kaplan-Meier method, Log rank test, and Cox proportional-hazards model analysis. NEK2 protein was overexpressed in malignant gliomas, but not in normal brain tissues. Overexpression of NEK2 correlated with malignancy, proliferation and adverse overall survival in gliomas. Moreover, chemotherapy, resection extent and WHO grade also correlate with overall survival in gliomas. However, within WHO grade II glioma subgroup, NEK2 overexpression showed no impact on overall survival. The present study firstly reveals that NEK2 protein is widely overexpressed in gliomas. NEK2 overexpression correlates significantly with malignancy (WHO grades), proliferation (Ki-67) and prognosis in malignant gliomas. NEK2 is a potential gene therapy target and prognostic indicator.

Prenatal cocaine exposure decreases parvalbumin-immunoreactive neurons and GABA-to-projection neuron ratio in the medial prefrontal cortex.

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McCarthy, Deirdre M; Bhide, Pradeep G

2012-01-01

Cocaine abuse during pregnancy produces harmful effects not only on the mother but also on the unborn child. The neurotransmitters dopamine and serotonin are known as the principal targets of the action of cocaine in the fetal and postnatal brain. However, recent evidence suggests that cocaine can impair cerebral cortical GABA neuron development and function. We sought to analyze the effects of prenatal cocaine exposure on the number and distribution of GABA and projection neurons (inhibitory interneurons and excitatory output neurons, respectively) in the mouse cerebral cortex. We found that the prenatal cocaine exposure decreased GABA neuron numbers and GABA-to-projection neuron ratio in the medial prefrontal cortex of 60-day-old mice. The neighboring prefrontal cortex did not show significant changes in either of these measures. However, there was a significant increase in projection neuron numbers in the prefrontal cortex but not in the medial prefrontal cortex. Thus, the effects of cocaine on GABA and projection neurons appear to be cortical region specific. The population of parvalbumin-immunoreactive GABA neurons was decreased in the medial prefrontal cortex following the prenatal cocaine exposure. The cocaine exposure also delayed the developmental decline in the volume of the medial prefrontal cortex. Thus, prenatal cocaine exposure produced persisting and region-specific effects on cortical cytoarchitecture and impaired the physiological balance between excitatory and inhibitory neurotransmission. These structural changes may underlie the electrophysiological and behavioral effects of prenatal cocaine exposure observed in animal models and human subjects. Copyright © 2012 S. Karger AG, Basel.

Overexpression of Glucocorticoid Receptor β Enhances Myogenesis and Reduces Catabolic Gene Expression.

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Hinds, Terry D; Peck, Bailey; Shek, Evan; Stroup, Steven; Hinson, Jennifer; Arthur, Susan; Marino, Joseph S

2016-02-11

Unlike the glucocorticoid receptor α (GRα), GR β (GRβ) has a truncated ligand-binding domain that prevents glucocorticoid binding, implicating GRα as the mediator of glucocorticoid-induced skeletal muscle loss. Because GRβ causes glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and dexamethasone (Dex) responsiveness. We measured GR isoform expression in C₂C12 muscle cells in response to Dex and insulin, and through four days of myotube formation. Next, lentiviral-mediated overexpression of GRβ in C₂C12 was performed, and these cells were characterized for cell fusion and myotube formation, as well as sensitivity to Dex via the expression of ubiquitin ligases. GRβ overexpression increased mRNA levels of muscle regulatory factors and enhanced proliferation in myoblasts. GRβ overexpressing myotubes had an increased fusion index. Myotubes overexpressing GRβ had lower forkhead box O3 (Foxo3a) mRNA levels and a blunted muscle atrophy F-box/Atrogen-1 (MAFbx) and muscle ring finger 1 (MuRF1) response to Dex. We showed that GRβ may serve as a pharmacological target for skeletal muscle growth and protection from glucocorticoid-induced catabolic signaling. Increasing GRβ levels in skeletal muscle may cause a state of glucocorticoid resistance, stabilizing muscle mass during exposure to high doses of glucocorticoids.

Overexpression of Glucocorticoid Receptor β Enhances Myogenesis and Reduces Catabolic Gene Expression

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Terry D. Hinds

2016-02-01

Full Text Available Unlike the glucocorticoid receptor α (GRα, GR β (GRβ has a truncated ligand-binding domain that prevents glucocorticoid binding, implicating GRα as the mediator of glucocorticoid-induced skeletal muscle loss. Because GRβ causes glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and dexamethasone (Dex responsiveness. We measured GR isoform expression in C2C12 muscle cells in response to Dex and insulin, and through four days of myotube formation. Next, lentiviral-mediated overexpression of GRβ in C2C12 was performed, and these cells were characterized for cell fusion and myotube formation, as well as sensitivity to Dex via the expression of ubiquitin ligases. GRβ overexpression increased mRNA levels of muscle regulatory factors and enhanced proliferation in myoblasts. GRβ overexpressing myotubes had an increased fusion index. Myotubes overexpressing GRβ had lower forkhead box O3 (Foxo3a mRNA levels and a blunted muscle atrophy F-box/Atrogen-1 (MAFbx and muscle ring finger 1 (MuRF1 response to Dex. We showed that GRβ may serve as a pharmacological target for skeletal muscle growth and protection from glucocorticoid-induced catabolic signaling. Increasing GRβ levels in skeletal muscle may cause a state of glucocorticoid resistance, stabilizing muscle mass during exposure to high doses of glucocorticoids.

Gene array analysis of PD-1H overexpressing monocytes reveals a pro-inflammatory profile

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Preeti Bharaj

2018-02-01

Full Text Available We have previously reported that overexpression of Programmed Death -1 Homolog (PD-1H in human monocytes leads to activation and spontaneous secretion of multiple pro inflammatory cytokines. Here we evaluate changes in monocytes gene expression after enforced PD-1H expression by gene array. The results show that there are significant alterations in 51 potential candidate genes that relate to immune response, cell adhesion and metabolism. Genes corresponding to pro-inflammatory cytokines showed the highest upregulation, 7, 3.2, 3.0, 5.8, 4.4 and 3.1 fold upregulation of TNF-α, IL-1 β, IFN-α, γ, λ and IL-27 relative to vector control. The data are in agreement with cytometric bead array analysis showing induction of proinflammatory cytokines, IL-6, IL-1β and TNF-α by PD-1H. Other genes related to inflammation, include transglutaminase 2 (TG2, NF-κB (p65 and p50 and toll like receptors (TLR 3 and 4 were upregulated 5, 4.5 and 2.5 fold, respectively. Gene set enrichment analysis (GSEA also revealed that signaling pathways related to inflammatory response, such as NFκB, AT1R, PYK2, MAPK, RELA, TNFR1, MTOR and proteasomal degradation, were significantly upregulated in response to PD-1H overexpression. We validated the results utilizing a standard inflammatory sepsis model in humanized BLT mice, finding that PD-1H expression was highly correlated with proinflammatory cytokine production. We therefore conclude that PD-1H functions to enhance monocyte activation and the induction of a pro-inflammatory gene expression profile.

ADAM12 overexpression does not improve outcome in mice with laminin alpha2-deficient muscular dystrophy

DEFF Research Database (Denmark)

Guo, Ling T; Shelton, G Diane; Wewer, Ulla M

2005-01-01

We have recently shown that overexpression of ADAM12 results in increased muscle regeneration and significantly reduced pathology in mdx, dystrophin deficient mice. In the present study, we tested the effect of overexpressing ADAM12 in dy(W) laminin-deficient mice. dy mice have a very severe...... clinical phenotype and would be expected to benefit greatly from enhanced regeneration. We found that dy(W) mice overexpressing ADAM12 indeed have increased muscle regeneration, as evidenced by increased numbers of muscle fibers expressing fetal myosin. However, overexpression of ADAM12 had no significant...

PRAME overexpression predicted good outcome in pediatric B-cell acute lymphoblastic leukemia patients receiving chemotherapy.

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Zhang, Yan-Huan; Lu, Ai-Dong; Yang, Lu; Li, Ling-Di; Chen, Wen-Min; Long, Ling-Yu; Zhang, Le-Ping; Qin, Ya-Zhen

2017-01-01

To investigate the prognostic value of PRAME expression in pediatric acute lymphoblastic leukemia(ALL), we measured PRAME transcript levels at diagnosis in 191 patients(146 B-ALL; 45T-ALL)receiving chemotherapy only. PRAME overexpression was defined as transcript levels higher than 0.30%, which is the upper limit of normal bone marrow and the optimal cutoff value derived from ROC curve analysis. PRAME overexpression was identified in 45.5% of patients. In B-ALL, PRAME overexpression was significantly associated with lower CIR(cumulative incidence of relapse), higher DFS (disease-freesurvival), and OS(overall survival) rates at 3 years, respectively (5.8% vs. 14.9%, P=0.014; 94.2% vs. 85.1%, P=0.014; 96.0% vs. 87.4%, P=0.039). PRAME overexpression had no impact on outcome in T-ALL patients. Among B-ALL patients with non-poor cytogenetic risk, those with PRAME overexpression showed significantly lower CIR, higher DFS and OS rates at 3 years, respectively (8.47% vs. 14.5%, P=0.009; 96.5% vs. 85.5%, P=0.009; 98.4% vs. 88.0%, P=0.023). Furthermore, PRAME overexpression was an independent good prognostic factor for relapse in all B-ALL patients and B-ALL patients with non-poor cytogenetic risk. Therefore, the prognostic significance of PRAME overexpression differed by ALL subtype; It predicted good outcome in pediatric B-ALL receiving chemotherapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Overexpression of protein O-fucosyltransferase 1 accelerates hepatocellular carcinoma progression via the Notch signaling pathway

International Nuclear Information System (INIS)

Ma, Lijie; Dong, Pingping; Liu, Longzi; Gao, Qiang; Duan, Meng; Zhang, Si; Chen, She; Xue, Ruyi; Wang, Xiaoying

2016-01-01

Aberrant activation of Notch signaling frequently occurs in liver cancer, and is associated with liver malignancies. However, the mechanisms regulating pathologic Notch activation in hepatocellular carcinoma (HCC) remain unclear. Protein O-fucosyltransferase 1 (Pofut1) catalyzes the addition of O-linked fucose to the epidermal growth factor-like repeats of Notch. In the present study, we detected the expression of Pofut1 in 8 HCC cell lines and 253 human HCC tissues. We reported that Pofut1 was overexpressed in HCC cell lines and clinical HCC tissues, and Pofut1 overexpression clinically correlated with the unfavorable survival and high disease recurrence in HCC. The in vitro assay demonstrated that Pofut1 overexpression accelerated the cell proliferation and migration in HCC cells. Furthermore, Pofut1 overexpression promoted the binding of Notch ligand Dll1 to Notch receptor, and hence activated Notch signaling pathway in HCC cells, indicating that Pofut1 overexpression could be a reason for the aberrant activation of Notch signaling in HCC. Taken together, our findings indicated that an aberrant activated Pofut1-Notch pathway was involved in HCC progression, and blockage of this pathway could be a promising strategy for the therapy of HCC. - Highlights: • Pofut1 overexpression in HCC was correlated with aggressive tumor behaviors. • Pofut1 overexpression in HCC was associated with poor prognosis. • Pofut1 promoted cell proliferation, migration and invasion in hepatoma cells. • Pofut1 activated Notch signaling pathway in hepatoma cells.

Overexpression of protein O-fucosyltransferase 1 accelerates hepatocellular carcinoma progression via the Notch signaling pathway

Energy Technology Data Exchange (ETDEWEB)

Ma, Lijie [Liver Surgery Department, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai (China); Dong, Pingping [Department of Gastroenterology and Hepatology, Shanghai Institute of Liver Diseases, Zhongshan Hospital of Fudan University, Shanghai (China); Liu, Longzi; Gao, Qiang; Duan, Meng [Liver Surgery Department, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai (China); Zhang, Si; Chen, She [Key Laboratory of Glycoconjugate Research Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Xue, Ruyi, E-mail: xue.ruyi@zs-hospital.sh.cn [Department of Gastroenterology and Hepatology, Shanghai Institute of Liver Diseases, Zhongshan Hospital of Fudan University, Shanghai (China); Wang, Xiaoying, E-mail: xiaoyingwang@fudan.edu.cn [Liver Surgery Department, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai (China)

2016-04-29

Aberrant activation of Notch signaling frequently occurs in liver cancer, and is associated with liver malignancies. However, the mechanisms regulating pathologic Notch activation in hepatocellular carcinoma (HCC) remain unclear. Protein O-fucosyltransferase 1 (Pofut1) catalyzes the addition of O-linked fucose to the epidermal growth factor-like repeats of Notch. In the present study, we detected the expression of Pofut1 in 8 HCC cell lines and 253 human HCC tissues. We reported that Pofut1 was overexpressed in HCC cell lines and clinical HCC tissues, and Pofut1 overexpression clinically correlated with the unfavorable survival and high disease recurrence in HCC. The in vitro assay demonstrated that Pofut1 overexpression accelerated the cell proliferation and migration in HCC cells. Furthermore, Pofut1 overexpression promoted the binding of Notch ligand Dll1 to Notch receptor, and hence activated Notch signaling pathway in HCC cells, indicating that Pofut1 overexpression could be a reason for the aberrant activation of Notch signaling in HCC. Taken together, our findings indicated that an aberrant activated Pofut1-Notch pathway was involved in HCC progression, and blockage of this pathway could be a promising strategy for the therapy of HCC. - Highlights: • Pofut1 overexpression in HCC was correlated with aggressive tumor behaviors. • Pofut1 overexpression in HCC was associated with poor prognosis. • Pofut1 promoted cell proliferation, migration and invasion in hepatoma cells. • Pofut1 activated Notch signaling pathway in hepatoma cells.

Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2.

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Alexander J Neumann

Full Text Available Articular cartilage progenitor cells (ACPCs represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs. This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2. hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1 concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase. To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs

Overexpressed KDM5B is associated with the progression of glioma and promotes glioma cell growth via downregulating p21

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Dai, Bin [Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038 (China); Hu, Zhiqiang, E-mail: zhiqhutg@126.com [Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038 (China); Huang, Hui; Zhu, Guangtong; Xiao, Zhiyong [Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038 (China); Wan, Weiqing; Zhang, Peng; Jia, Wang; Zhang, Liwei [Department of Neurosurgery, Beijing Tian Tan Hospital, Capital Medical University, Beijing 100050 (China)

2014-11-07

Highlights: • KDM5B is overexpressed in glioma samples. • KDM5B stimulated proliferation of glioma cells. • Inhibition of p21contributes to KDM5B-induced proliferation. - Abstract: Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Upregulation of lysine (K)-specific demethylase 5B (KDM5B) has been reported in a variety of malignant tumors. However, the impact of KDM5B in glioma remains unclear. The objective of this study was to investigate the expression and prognostic value of KDM5B in glioma. In clinical glioma samples, we found that KDM5B expression was significantly upregulated in cancer lesions compared with normal brain tissues. Kaplan–Meier analysis showed that patients with glioma and higher KDM5B expression tend to have shorter overall survival time. By silencing or overexpressing KDM5B in glioma cells, we found that KDM5B could promote cell growth both in vitro and in vivo. Moreover, we demonstrated that KDM5B promoted glioma proliferation partly via regulation of the expression of p21. Our study provided evidence that KDM5B functions as a novel tumor oncogene in glioma and may be a potential therapeutic target for glioma management.

An overexpression of chalcone reductase of Pueraria montana var. lobata alters biosynthesis of anthocyanin and 5'-deoxyflavonoids in transgenic tobacco.

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Joung, Jae-youl; Kasthuri, G Mangai; Park, Ji-young; Kang, Won-jin; Kim, Hyun-soon; Yoon, Bong-sik; Joung, Hyouk; Jeon, Jae-heung

2003-03-28

We isolated the chalcone reductase (pl-chr) gene of Pueraria montana var. lobata by using a PCR strategy from cDNA pools of storage roots. A high level of expression of RNA was found in both stems and roots. The genomic Southern blot result suggests that pl-chr exists as a member of a small gene family. By introducing a pl-chr gene under the control of the 35S CaMV promoter into the pink-flowering Xanthi line of Nicotiana tabacum, the flower color was changed from pink to white-to-pink. The contents of anthocyanin in the flowers of the transgenic lines were dramatically decreased by 40%, but the total UV absorption compounds remained unchanged. The production of liquiritigenin in pl-chr overexpressed transgenic tobacco lines was confirmed by HPLC and MS analysis. The introduction of pl-chr gene provides a method to redirect the flavonoid pathway into 5'-deoxyflavonoid production in non-legume crops, in order to manipulate the phenylpropanoid pathway for isoflavonoid production.

Overexpression of Oct4 suppresses the metastatic potential of breast cancer cells via Rnd1 downregulation.

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Shen, Long; Qin, Kunhua; Wang, Dekun; Zhang, Yan; Bai, Nan; Yang, Shengyong; Luo, Yunping; Xiang, Rong; Tan, Xiaoyue

2014-11-01

Although Oct4 is known as a critical transcription factor involved in maintaining "stemness", its role in tumor metastasis is still controversial. Herein, we overexpressed and silenced Oct4 expression in two breast cancer cell lines, MDA-MB-231 and 4T1, separately. Our data showed that ectopic overexpression of Oct4 suppressed cell migration and invasion in vitro and the formation of metastatic lung nodules in vivo. Conversely, Oct4 downregulation increased the metastatic potential of breast cancer cells both in vitro and in vivo. Furthermore, we identified Rnd1 as the downstream target of Oct4 by ribonucleic acid sequencing (RNA-seq) analysis, which was significantly downregulated upon Oct4 overexpression. Chromatin immunoprecipitation assays revealed the binding of Oct4 to the promoter region of Rnd1 by ectopic overexpression of Oct4. Dual luciferase assays indicated that Oct4 overexpression suppressed transcriptional activity of the Rnd1 promoter. Moreover, overexpression of Rnd1 partially rescued the inhibitory effects of Oct4 on the migration and invasion of breast cancer cells. Overexpression of Rnd1 counteracted the influence of Oct4 on the formation of cell adhesion and lamellipodia, which implied a potential underlying mechanism involving Rnd1. In addition, we also found that overexpression of Oct4 led to an elevation of E-cadherin expression, even in 4T1 cells that possess a relatively high basal level of E-cadherin. Rnd1 overexpression impaired the promoting effects of Oct4 on E-cadherin expression in MDA-MB-231 cells. These results suggest that Oct4 affects the metastatic potential of breast cancer cells through Rnd1-mediated effects that influence cell motility and E-cadherin expression. Copyright © 2014 Elsevier B.V. All rights reserved.

L-Endoglin Overexpression Increases Renal Fibrosis after Unilateral Ureteral Obstruction

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Arévalo, Miguel; Núñez-Gómez, Elena; Pérez-Roque, Lucía; Pericacho, Miguel; González-Núñez, María; Langa, Carmen; Martínez-Salgado, Carlos; Perez-Barriocanal, Fernando; Bernabeu, Carmelo; Lopez-Novoa, José M.

2014-01-01

Transforming growth factor-β (TGF-β) plays a pivotal role in renal fibrosis. Endoglin, a 180 KDa membrane glycoprotein, is a TGF-β co-receptor overexpressed in several models of chronic kidney disease, but its function in renal fibrosis remains uncertain. Two membrane isoforms generated by alternative splicing have been described, L-Endoglin (long) and S-Endoglin (short) that differ from each other in their cytoplasmic tails, being L-Endoglin the most abundant isoform. The aim of this study was to assess the effect of L-Endoglin overexpression in renal tubulo-interstitial fibrosis. For this purpose, a transgenic mouse which ubiquitously overexpresses human L-Endoglin (L-ENG+) was generated and unilateral ureteral obstruction (UUO) was performed in L-ENG+ mice and their wild type (WT) littermates. Obstructed kidneys from L-ENG+ mice showed higher amounts of type I collagen and fibronectin but similar levels of α-smooth muscle actin (α-SMA) than obstructed kidneys from WT mice. Smad1 and Smad3 phosphorylation were significantly higher in obstructed kidneys from L-ENG+ than in WT mice. Our results suggest that the higher increase of renal fibrosis observed in L-ENG+ mice is not due to a major abundance of myofibroblasts, as similar levels of α-SMA were observed in both L-ENG+ and WT mice, but to the higher collagen and fibronectin synthesis by these fibroblasts. Furthermore, in vivo L-Endoglin overexpression potentiates Smad1 and Smad3 pathways and this effect is associated with higher renal fibrosis development. PMID:25313562

Overexpressed Genes/ESTs and Characterization of Distinct Amplicons on 17823 in Breast Cancer Cells

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Ayse E. Erson

2001-01-01

Full Text Available 17823 is a frequent site of gene amplification in breast cancer. Several lines of evidence suggest the presence of multiple amplicons on 17823. To characterize distinct amplicons on 17823 and localize putative oncogenes, we screened genes and expressed sequence tags (ESTs in existing physical and radiation hybrid maps for amplification and overexpression in breast cancer cell lines by semiquantitative duplex PCR, semiquantitative duplex RT-PCR, Southern blot, Northern blot analyses. We identified two distinct amplicons on 17823, one including TBX2 and another proximal region including RPS6KB1 (PS6K and MUL. In addition to these previously reported overexpressed genes, we also identified amplification and overexpression of additional uncharacterized genes and ESTs, some of which suggest potential oncogenic activity. In conclusion, we have further defined two distinct regions of gene amplification and overexpression on 17823 with identification of new potential oncogene candidates. Based on the amplification and overexpression patterns of known and as of yet unrecognized genes on 17823, it is likely that some of these genes mapping to the discrete amplicons function as oncogenes and contribute to tumor progression in breast cancer cells.

Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

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Mattanovich Diethard

2008-07-01

Full Text Available Abstract Background High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes. Results Overexpression of the first gene of the riboflavin biosynthetic pathway (RIB1 is already sufficient to obtain yellow colonies and the accumulation of riboflavin in the supernatant of shake flask cultures growing on glucose. Sequential deregulation of all the genes, by exchange of their native promoter with the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP increases the riboflavin accumulation significantly. Conclusion The regulation of the pathway is distributed over more than one gene. High cell density cultivations of a P. pastoris strain overexpressing all six RIB genes allow the accumulation of 175 mg/L riboflavin in the supernatant. The basis for rational engineering of riboflavin production in P. pastoris has thus been established.

Targeted overexpression of mitochondrial catalase protects against cancer chemotherapy-induced skeletal muscle dysfunction.

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Gilliam, Laura A A; Lark, Daniel S; Reese, Lauren R; Torres, Maria J; Ryan, Terence E; Lin, Chien-Te; Cathey, Brook L; Neufer, P Darrell

2016-08-01

The loss of strength in combination with constant fatigue is a burden on cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and increases mitochondrial H2O2 We hypothesized that the combined effect of cancer and chemotherapy in an immunocompetent breast cancer mouse model (E0771) would compromise skeletal muscle mitochondrial respiratory function, leading to an increase in H2O2-emitting potential and impaired muscle function. Here, we demonstrate that cancer chemotherapy decreases mitochondrial respiratory capacity supported with complex I (pyruvate/glutamate/malate) and complex II (succinate) substrates. Mitochondrial H2O2-emitting potential was altered in skeletal muscle, and global protein oxidation was elevated with cancer chemotherapy. Muscle contractile function was impaired following exposure to cancer chemotherapy. Genetically engineering the overexpression of catalase in mitochondria of muscle attenuated mitochondrial H2O2 emission and protein oxidation, preserving mitochondrial and whole muscle function despite cancer chemotherapy. These findings suggest mitochondrial oxidants as a mediator of cancer chemotherapy-induced skeletal muscle dysfunction. Copyright © 2016 the American Physiological Society.

Mediobasal hypothalamic overexpression of DEPTOR protects against high-fat diet-induced obesity

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Alexandre Caron

2016-02-01

Full Text Available Background/Objective: The mechanistic target of rapamycin (mTOR is a serine–threonine kinase that functions into distinct protein complexes (mTORC1 and mTORC2 that regulate energy homeostasis. DEP-domain containing mTOR-interacting protein (DEPTOR is part of these complexes and is known to dampen mTORC1 function, consequently reducing mTORC1 negative feedbacks and promoting insulin signaling and Akt/PKB activation in several models. Recently, we observed that DEPTOR is expressed in several structures of the brain including the mediobasal hypothalamus (MBH, a region that regulates energy balance. Whether DEPTOR in the MBH plays a functional role in regulating energy balance and hypothalamic insulin signaling has never been tested. Methods: We have generated a novel conditional transgenic mouse model based on the Cre-LoxP system allowing targeted overexpression of DEPTOR. Mice overexpressing DEPTOR in the MBH were subjected to a metabolic phenotyping and MBH insulin signaling was evaluated. Results: We first report that systemic (brain and periphery overexpression of DEPTOR prevents high-fat diet-induced obesity, improves glucose metabolism and protects against hepatic steatosis. These phenotypes were associated with a reduction in food intake and feed efficiency and an elevation in oxygen consumption. Strikingly, specific overexpression of DEPTOR in the MBH completely recapitulated these phenotypes. DEPTOR overexpression was associated with an increase in hypothalamic insulin signaling, as illustrated by elevated Akt/PKB activation. Conclusion: Altogether, these results support a role for MBH DEPTOR in the regulation of energy balance and metabolism. Keywords: mTOR, DEPTOR, Hypothalamus, Energy balance, Glucose metabolism

The Effects of NDRG2 Overexpression on Cell Proliferation and Invasiveness of SW48 Colorectal Cancer Cell Line.

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Golestan, Ali; Mojtahedi, Zahra; Ghalamfarsa, Ghasem; Hamidinia, Maryam; Takhshid, Mohammad Ali

2015-09-01

Colorectal cancer (CRC) is one of the most common causes of cancer-related death in the world. The expression of N-myc downstream-regulated gene 2 (NDRG2) is down-regulated in CRC. The aim of this study was to investigate the effect of NDRG2 overexpression on cell proliferation and invasive potential of SW48 cells. SW48 cells were transfected with a plasmid overexpressing NDRG2. After stable transfection, the effect of NDRG2 overexpression on cell proliferation was evaluated by MTT assay. The effects of NDRG2 overexpression on cell migration, invasion and cell motility and matrix metalloproteinase 9 (MMP9) activities were also investigated using matrigel transwell assay, wound healing assay and gelatin zymography, respectively. MTT assay showed that overexpression of NDRG2 caused attenuation of SW48 cell proliferation. Transwell and wound healing assay revealed that NDRG2 overexpression led to inhibition of migration, invasion, and motility of SW48 cells. The overexpression of NDRG2 also reduced the activity of secreted MMP-9. The results of this study suggest that NDRG2 overexpression inhibits proliferation and invasive potential of SW48 cells, which likely occurs via suppression of MMP-9 activity.

Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis.

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Zhang, Chunyu; Zhang, Wei; Ren, Guodong; Li, Delin; Cahoon, Rebecca E; Chen, Ming; Zhou, Yongming; Yu, Bin; Cahoon, Edgar B

2015-08-01

Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis. © 2015 American Society of

Dek overexpression in murine epithelia increases overt esophageal squamous cell carcinoma incidence

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Cimperman, Katherine A.; Haas, Sarah R.; Guasch, Geraldine; Waclaw, Ronald R.; Komurov, Kakajan; Lane, Adam; Wikenheiser-Brokamp, Kathryn A.

2018-01-01

Esophageal cancer occurs as either squamous cell carcinoma (ESCC) or adenocarcinoma. ESCCs comprise almost 90% of cases worldwide, and recur with a less than 15% five-year survival rate despite available treatments. The identification of new ESCC drivers and therapeutic targets is critical for improving outcomes. Here we report that expression of the human DEK oncogene is strongly upregulated in esophageal SCC based on data in the cancer genome atlas (TCGA). DEK is a chromatin-associated protein with important roles in several nuclear processes including gene transcription, epigenetics, and DNA repair. Our previous data have utilized a murine knockout model to demonstrate that Dek expression is required for oral and esophageal SCC growth. Also, DEK overexpression in human keratinocytes, the cell of origin for SCC, was sufficient to cause hyperplasia in 3D organotypic raft cultures that mimic human skin, thus linking high DEK expression in keratinocytes to oncogenic phenotypes. However, the role of DEK over-expression in ESCC development remains unknown in human cells or genetic mouse models. To define the consequences of Dek overexpression in vivo, we generated and validated a tetracycline responsive Dek transgenic mouse model referred to as Bi-L-Dek. Dek overexpression was induced in the basal keratinocytes of stratified squamous epithelium by crossing Bi-L-Dek mice to keratin 5 tetracycline transactivator (K5-tTA) mice. Conditional transgene expression was validated in the resulting Bi-L-Dek_K5-tTA mice and was suppressed with doxycycline treatment in the tetracycline-off system. The mice were subjected to an established HNSCC and esophageal carcinogenesis protocol using the chemical carcinogen 4-nitroquinoline 1-oxide (4NQO). Dek overexpression stimulated gross esophageal tumor development, when compared to doxycycline treated control mice. Furthermore, high Dek expression caused a trend toward esophageal hyperplasia in 4NQO treated mice. Taken together, these

Changes in Otx2 and Parvalbumin Immunoreactivity in the Superior Colliculus in the Platelet-Derived Growth Factor Receptor-β Knockout Mice

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Juanjuan Zhao

2013-01-01

Full Text Available The superior colliculus (SC, a relay nucleus in the subcortical visual pathways, is implicated in socioemotional behaviors. Homeoprotein Otx2 and β subunit of receptors of platelet-derived growth factor (PDGFR-β have been suggested to play an important role in development of the visual system and development and maturation of GABAergic neurons. Although PDGFR-β-knockout (KO mice displayed socio-emotional deficits associated with parvalbumin (PV-immunoreactive (IR neurons, their anatomical bases in the SC were unknown. In the present study, Otx2 and PV-immunolabeling in the adult mouse SC were investigated in the PDGFR-β KO mice. Although there were no differences in distribution patterns of Otx2 and PV-IR cells between the wild type and PDGFR-β KO mice, the mean numbers of both of the Otx2- and PV-IR cells were significantly reduced in the PDGFR-β KO mice. Furthermore, average diameters of Otx2- and PV-IR cells were significantly reduced in the PDGFR-β KO mice. These findings suggest that PDGFR-β plays a critical role in the functional development of the SC through its effects on Otx2- and PV-IR cells, provided specific roles of Otx2 protein and PV-IR cells in the development of SC neurons and visual information processing, respectively.

Clinical importance of TERT overexpression in hepatocellular carcinoma treated with curative surgical resection in HBV endemic area.

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Yu, Jeong Il; Choi, Changhoon; Ha, Sang Yun; Park, Cheol-Keun; Kang, So Young; Joh, Jae-Won; Paik, Seung Woon; Kim, Seonwoo; Kim, Minji; Jung, Sang Hoon; Park, Hee Chul

2017-09-25

This study was designed to investigate the associations between TERT overexpression and the clinicopathologic factors of hepatocellular carcinoma (HCC). A total of 291 patients with HCC were enrolled. The site of first recurrence (anywhere in the liver) was classified as intrahepatic recurrence (IHR). Recurrence was then sub classified as either early or late IHR according to whether it was discovered within 2 years of resection, or after, respectively. TERT overexpression was not significantly correlated with previously recognized prognostic factors. During follow-up, early IHR occurred in 126 (63.6%) patients, while late IHR was detected in 59 patients among 145 patients who remained free of HCC recurrence for ≥ 2 years after surgery. Multivariate analysis showed late IHR was significantly correlated with TERT overexpression (P overexpression (P overexpression was the only significant prognostic factor for late IHR in HCC treated with curative resection. And, the statistical significance of TERT overexpression on late IHR was limited to HBsAg-positive patients.

Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone

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Sanmartin, Maite; Drogoudi, Pavlina D.; Lyons, Tom

2003-01-01

overexpressing plants exposed to 100 nmol mol-1 ozone for 7 h day-1 exhibited a substantial increase in foliar injury, and a greater pollutant-induced reduction in both the light-saturated rate of CO2 assimilation and the maximum in vivo rate of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation......Transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants expressing cucumber ascorbate oxidase (EC.1.10.3.3) were used to examine the role of extracellular ascorbic acid in mediating tolerance to the ubiquitous air pollutant, ozone (O3). Three homozygous transgenic lines, chosen on the basis...

[Characteristic and clinical significance of DNA methyltransferase 3B overexpression in endometrial carcinoma].

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Dong, Y; Zhou, M; Ba, X J; Si, J W; Li, W T; Wang, Y; Li, D; Li, T

2016-10-18

To determine the clinicopathological significance of the DNA methyltransferase 3B (DNMT3B) overexpression in endometrial carcinomas and to evaluate its correlation with hormone receptor status. Immunohistochemistry was performed to assess the expression of DNMT3B and hormone receptors in 104 endometrial carcinomas. DNMT3B overexpression occurred frequently in endometrioid carcinoma (EC, 54.8%) more than in nonendometrioid carcinoma (NEC, 30.0%) with statistical significance (P=0.028). Furthermore, there was a trend that EC with worse clinico-pathological variables and shorter survival had a higher DNMT3B expression, and the correlation between DNMT3B and tumor grade reached statistical significance (P=0.019).A negative correlation between DNMT3B and estrogen receptor (ER) or progesterone receptor (PR) expression was found in EC. NMT3B overexpression occurred frequently in the ER or PR negative subgroups (78.9%, 86.7%) more than in the positive subgroups (47.7%, 47.8%) with statistical significance (P=0.016, P=0.006). In addition, the DNMT3B overexpression increased in tumors with both ER and PR negative expression (92.9%, P=0.002). However, no such correlation was found in NEC (P>0.05). Sequence analyses demonstrated multiple ER and PR binding sites in the promoter regions of DNMT3B gene. This study showed that the expression of DNMT3B in EC and NEC was different. DNMT3B overexpression in EC was associated with the worse clinicopathological variables and might have predictive value. The methylation status of EC and NEC maybe different. In addition, in EC, DNMT3B overexpression negatively correlated with ER or PR expression. In NEC, the correlation between DNMT3B and ER or PR status was not present.

genetic overexpression of NR2B subunit enhances social recognition memory for different strains and species.

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Jacobs, Stephanie A; Tsien, Joe Z

2012-01-01

The ability to learn and remember conspecifics is essential for the establishment and maintenance of social groups. Many animals, including humans, primates and rodents, depend on stable social relationships for survival. Social learning and social recognition have become emerging areas of interest for neuroscientists but are still not well understood. It has been established that several hormones play a role in the modulation of social recognition including estrogen, oxytocin and arginine vasopression. Relatively few studies have investigated how social recognition might be improved or enhanced. In this study, we investigate the role of the NMDA receptor in social recognition memory, specifically the consequences of altering the ratio of the NR2B:NR2A subunits in the forebrain regions in social behavior. We produced transgenic mice in which the NR2B subunit of the NMDA receptor was overexpressed postnatally in the excitatory neurons of the forebrain areas including the cortex, amygdala and hippocampus. We investigated the ability of both our transgenic animals and their wild-type littermate to learn and remember juvenile conspecifics using both 1-hr and 24-hr memory tests. Our experiments show that the wild-type animals and NR2B transgenic mice preformed similarly in the 1-hr test. However, transgenic mice showed better performances in 24-hr tests of recognizing animals of a different strain or animals of a different species. We conclude that NR2B overexpression in the forebrain enhances social recognition memory for different strains and animal species.

Opposite effect of phencyclidine on activity-regulated cytoskeleton-associated protein (Arc) in juvenile and adult limbic rat brain regions

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Thomsen, Morten S; Hansen, Henrik H; Mikkelsen, Jens D

2010-01-01

-regulated cytoskeleton-associated protein (Arc) and parvalbumin mRNA expression in juvenile and adult rats. Arc is a marker for excitatory neurotransmission. Parvalbumin is a marker for GABAergic neurotransmission, known to be reduced in postmortem brains of schizophrenics. PCP reduced parvalbumin mRNA expression...

Prediction of recombinant protein overexpression in Escherichia coli using a machine learning based model (RPOLP).

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Habibi, Narjeskhatoon; Norouzi, Alireza; Mohd Hashim, Siti Z; Shamsir, Mohd Shahir; Samian, Razip

2015-11-01

Recombinant protein overexpression, an important biotechnological process, is ruled by complex biological rules which are mostly unknown, is in need of an intelligent algorithm so as to avoid resource-intensive lab-based trial and error experiments in order to determine the expression level of the recombinant protein. The purpose of this study is to propose a predictive model to estimate the level of recombinant protein overexpression for the first time in the literature using a machine learning approach based on the sequence, expression vector, and expression host. The expression host was confined to Escherichia coli which is the most popular bacterial host to overexpress recombinant proteins. To provide a handle to the problem, the overexpression level was categorized as low, medium and high. A set of features which were likely to affect the overexpression level was generated based on the known facts (e.g. gene length) and knowledge gathered from related literature. Then, a representative sub-set of features generated in the previous objective was determined using feature selection techniques. Finally a predictive model was developed using random forest classifier which was able to adequately classify the multi-class imbalanced small dataset constructed. The result showed that the predictive model provided a promising accuracy of 80% on average, in estimating the overexpression level of a recombinant protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

IMPAIRED PROCESSING IN THE PRIMARY AUDITORY CORTEX OF AN ANIMAL MODEL OF AUTISM

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Renata eAnomal

2015-11-01

Full Text Available Autism is a neurodevelopmental disorder clinically characterized by deficits in communication, lack of social interaction and, repetitive behaviors with restricted interests. A number of studies have reported that sensory perception abnormalities are common in autistic individuals and might contribute to the complex behavioral symptoms of the disorder. In this context, hearing incongruence is particularly prevalent. Considering that some of this abnormal processing might stem from the unbalance of inhibitory and excitatory drives in brain circuitries, we used an animal model of autism induced by valproic acid (VPA during pregnancy in order to investigate the tonotopic organization of the primary auditory cortex (AI and its local inhibitory circuitry. Our results show that VPA rats have distorted primary auditory maps with over-representation of high frequencies, broadly tuned receptive fields and higher sound intensity thresholds as compared to controls. However, we did not detect differences in the number of parvalbumin-positive interneurons in AI of VPA and control rats. Altogether our findings show that neurophysiological impairments of hearing perception in this autism model occur independently of alterations in the number of parvalbumin-expressing interneurons. These data support the notion that fine circuit alterations, rather than gross cellular modification, could lead to neurophysiological changes in the autistic brain.

Overexpression of CHMP7 from rapeseed and Arabidopsis causes dwarfism and premature senescence in Arabidopsis.

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Yang, Hongli; Liu, Jing; Lin, Jiulu; Deng, Linbin; Fan, Shihang; Guo, Yan; Sun, Fengming; Hua, Wei

2016-10-01

Endosomal sorting complexes required for transport (ESCRT) are well known in mammalians and yeast and plays an essential role in the formation of multi-vesicular bodies. Accumulating evidence has shown that ESCRT proteins contribute to proper plant development. CHMP7 (charged multi-vesicular body protein 7) is an ESCRT-III-related protein and functions in the endosomal sorting pathway in humans. However, its function in plants has not been explored in detail. In this study, we isolate the putative homolog of CHMP7 from rapeseed, BnCHMP7, which contains eight exons and encodes a protein consisting of 423 amino acid residues. Compared with the wild-type, overexpression of BnCHMP7 in Arabidopsis disturbs plant growth and decreases seed yield. Moreover, the transgenic plants also display early leaf senescence and hypersensitivity to dark treatment due to defects in autophagic degradation. Further study showed that BnCHMP7 is highly expressed in leaves and that YFP-BnCHMP7 is predominantly localized in endosome. Compared with human CHMP7, we found that BnCHMP7 not only interacts with ESCRT-III subunits SNF7.2 (CHMP4B), but also with VPS2.2 and CHMP1B. As expected, microarray analysis revealed that the expression of ESCRT transport genes is significantly affected. Additionally, the expression of some genes that are involved in senescence, protein synthesis and protein degradation is also altered in BnCHMP7-overexpressing plants. Taken together, BnCHMP7 encodes an endosome-localized protein, which causes dwarfism and leaf senescence as an ESCRT-III-related component. Copyright © 2016 Elsevier GmbH. All rights reserved.

Overexpression of HDAC1 induces cellular senescence by Sp1/PP2A/pRb pathway

International Nuclear Information System (INIS)

Chuang, Jian-Ying; Hung, Jan-Jong

2011-01-01

Highlights: → Overexpression of HDAC1 induces Sp1 deacetylation and raises Sp1/p300 complex formation to bind to PP2Ac promoter. → Overexpression of HDAC1 strongly inhibits the phosphorylation of pRb through up-regulation of PP2A. → Overexpressed HDAC1 restrains cell proliferaction and induces cell senescence though a novel Sp1/PP2A/pRb pathway. -- Abstract: Senescence is associated with decreased activities of DNA replication, protein synthesis, and cellular division, which can result in deterioration of cellular functions. Herein, we report that the growth and division of tumor cells were significantly repressed by overexpression of histone deacetylase (HDAC) 1 with the Tet-off induced system or transient transfection. In addition, HDAC1 overexpression led to senescence through both an accumulation of hypophosphorylated active retinoblastoma protein (pRb) and an increase in the protein level of protein phosphatase 2A catalytic subunit (PP2Ac). HDAC1 overexpression also increased the level of Sp1 deacetylation and elevated the interaction between Sp1 and p300, and subsequently that Sp1/p300 complex bound to the promoter of PP2Ac, thus leading to induction of PP2Ac expression. Similar results were obtained in the HDAC1-Tet-off stable clone. Taken together, these results indicate that HDAC1 overexpression restrained cell proliferation and induced premature senescence in cervical cancer cells through a novel Sp1/PP2A/pRb pathway.

Overexpression of HDAC1 induces cellular senescence by Sp1/PP2A/pRb pathway

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Chuang, Jian-Ying [Department of Pharmacology, National Cheng-Kung University, Tainan 701, Taiwan (China); Hung, Jan-Jong, E-mail: petehung@mail.ncku.edu.tw [Department of Pharmacology, National Cheng-Kung University, Tainan 701, Taiwan (China); Institute of Bioinformatics and Biosignal Transduction, National Cheng-Kung University, Tainan 701, Taiwan (China)

2011-04-15

Highlights: {yields} Overexpression of HDAC1 induces Sp1 deacetylation and raises Sp1/p300 complex formation to bind to PP2Ac promoter. {yields} Overexpression of HDAC1 strongly inhibits the phosphorylation of pRb through up-regulation of PP2A. {yields} Overexpressed HDAC1 restrains cell proliferaction and induces cell senescence though a novel Sp1/PP2A/pRb pathway. -- Abstract: Senescence is associated with decreased activities of DNA replication, protein synthesis, and cellular division, which can result in deterioration of cellular functions. Herein, we report that the growth and division of tumor cells were significantly repressed by overexpression of histone deacetylase (HDAC) 1 with the Tet-off induced system or transient transfection. In addition, HDAC1 overexpression led to senescence through both an accumulation of hypophosphorylated active retinoblastoma protein (pRb) and an increase in the protein level of protein phosphatase 2A catalytic subunit (PP2Ac). HDAC1 overexpression also increased the level of Sp1 deacetylation and elevated the interaction between Sp1 and p300, and subsequently that Sp1/p300 complex bound to the promoter of PP2Ac, thus leading to induction of PP2Ac expression. Similar results were obtained in the HDAC1-Tet-off stable clone. Taken together, these results indicate that HDAC1 overexpression restrained cell proliferation and induced premature senescence in cervical cancer cells through a novel Sp1/PP2A/pRb pathway.

Cortical deficits of glutamic acid decarboxylase 67 expression in schizophrenia: clinical, protein, and cell type-specific features.

Science.gov (United States)

Curley, Allison A; Arion, Dominique; Volk, David W; Asafu-Adjei, Josephine K; Sampson, Allan R; Fish, Kenneth N; Lewis, David A

2011-09-01

Cognitive deficits in schizophrenia are associated with altered activity of the dorsolateral prefrontal cortex, which has been attributed to lower expression of the 67 kDa isoform of glutamic acid decarboxylase (GAD67), the major γ-aminobutyric acid (GABA)-synthesizing enzyme. However, little is known about the relationship of prefrontal GAD67 mRNA levels and illness severity, translation of the transcript into protein, and protein levels in axon terminals, the key site of GABA production and function. Quantitative polymerase chain reaction was used to measure GAD67 mRNA levels in postmortem specimens of dorsolateral prefrontal cortex from subjects with schizophrenia and matched comparison subjects with no known history of psychiatric or neurological disorders (N=42 pairs). In a subset of this cohort in which potential confounds of protein measures were controlled (N=19 pairs), Western blotting was used to quantify tissue levels of GAD67 protein in tissue. In five of these pairs, multilabel confocal immunofluorescence was used to quantify GAD67 protein levels in the axon terminals of parvalbumin-containing GABA neurons, which are known to have low levels of GAD67 mRNA in schizophrenia. GAD67 mRNA levels were significantly lower in schizophrenia subjects (by 15%), but transcript levels were not associated with predictors or measures of illness severity or chronicity. In schizophrenia subjects, GAD67 protein levels were significantly lower in total gray matter (by 10%) and in parvalbumin axon terminals (by 49%). The findings that lower GAD67 mRNA expression is common in schizophrenia, that it is not a consequence of having the illness, and that it leads to less translation of the protein, especially in the axon terminals of parvalbumin-containing neurons, support the hypothesis that lower GABA synthesis in parvalbumin neurons contributes to dorsolateral prefrontal cortex dysfunction and impaired cognition in schizophrenia.

Parvalbumin-expressing ependymal cells in rostral lateral ventricle wall adhesions contribute to aging-related ventricle stenosis in mice.

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Filice, Federica; Celio, Marco R; Babalian, Alexandre; Blum, Walter; Szabolcsi, Viktoria

2017-10-15

Aging-associated ependymal-cell pathologies can manifest as ventricular gliosis, ventricle enlargement, or ventricle stenosis. Ventricle stenosis and fusion of the lateral ventricle (LV) walls is associated with a massive decline of the proliferative capacities of the stem cell niche in the affected subventricular zone (SVZ) in aging mice. We examined the brains of adult C57BL/6 mice and found that ependymal cells located in the adhesions of the medial and lateral walls of the rostral LVs upregulated parvalbumin (PV) and displayed reactive phenotype, similarly to injury-reactive ependymal cells. However, PV+ ependymal cells in the LV-wall adhesions, unlike injury-reactive ones, did not express glial fibrillary acidic protein. S100B+/PV+ ependymal cells found in younger mice diminished in the LV-wall adhesions throughout aging. We found that periventricular PV-immunofluorescence showed positive correlation to the grade of LV stenosis in nonaged mice (wall adhesions and LV stenosis was significantly lower in mid-aged (>10-month-old) PV-knock out (PV-KO) mice. This suggests an involvement of PV+ ependymal cells in aging-associated ventricle stenosis. Additionally, we observed a time-shift in microglial activation in the LV-wall adhesions between age-grouped PV-KO and wild-type mice, suggesting a delay in microglial activation when PV is absent from ependymal cells. Our findings implicate that compromised ependymal cells of the adhering ependymal layers upregulate PV and display phenotype shift to "reactive" ependymal cells in aging-related ventricle stenosis; moreover, they also contribute to the progression of LV-wall fusion associated with a decline of the affected SVZ-stem cell niche in aged mice. © 2017 Wiley Periodicals, Inc.

Targeted Transgenic Overexpression of Mitochondrial Thymidine Kinase (TK2) Alters Mitochondrial DNA (mtDNA) and Mitochondrial Polypeptide Abundance

Science.gov (United States)

Hosseini, Seyed H.; Kohler, James J.; Haase, Chad P.; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-01-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-γ. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity. PMID:17322372

Role of overexpressed CFA/I fimbriae in bacterial swimming

International Nuclear Information System (INIS)

Cao, Ling; Lim, Timothy; Jun, SangMu; Riccardi, Carol; Yang, Xinghong; Suo, Zhiyong; Deliorman, Muhammedin; Kellerman, Laura; Avci, Recep

2012-01-01

Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility. (paper)

Matrix metalloproteinase-8 overexpression prevents proper tissue repair

DEFF Research Database (Denmark)

Danielsen, Patricia L; Holst, Anders V; Maltesen, Henrik R

2011-01-01

The collagenolytic matrix metalloproteinase-8 (MMP-8) is essential for normal tissue repair but is often overexpressed in wounds with disrupted healing. Our aim was to study the impact of a local excess of this neutrophil-derived proteinase on wound healing using recombinant adenovirus...

Overexpression and RNA interference of TwDXR regulate the accumulation of terpenoid active ingredients in Tripterygium wilfordii.

Science.gov (United States)

Zhang, Yifeng; Zhao, Yujun; Wang, Jiadian; Hu, Tianyuan; Tong, Yuru; Zhou, Jiawei; Song, Yadi; Gao, Wei; Huang, Luqi

2018-02-01

To examine the putative regulatory role of TwDXR in terpenoid biosynthesis and terpenoid biosynthetic pathway-related gene expression, through overexpression and RNA interference with TwDXR. We obtained 1410 and 454 bp TwDXR-specific fragments to construct overexpression and RNAi vectors. qRT-PCR was used to detect the expression of TwDXR and terpenoid biosynthesis pathway-related genes. The overexpression of TwDXR led to a 285% upregulation and the TwDXR RNAi led to a reduction to 26% of the control (empty vector-transformed cells) levels. However, pathway-related genes displayed different trends. When TwDXR was overexpressed, TwDXS expression decreased by 31% but increased to 198% when TwDXR expression was inhibited. The accumulation of terpenoids was also assayed. In the overexpression group, differences were not significant whereas the contents of triptolide and celastrol in the TwDXR RNAi samples were diminished by 27.3 and 24.0%, respectively. The feedback regulation of gene transcription and the accumulation of terpenoids in terpenoid biosynthesis in Tripterygium wilfordii were verified by TwDXR overexpression and RNAi experiments.

High eIF4E Overexpression in Node Negative Breast Cancer as Predictor for Recurrence

National Research Council Canada - National Science Library

Li, Benjamin

2002-01-01

.... EIF4E overexpression has been found in human malignancies (Li, 1997; Nathan, 1997). Furthermore, there appears to be an association of eIF4E overexpression and clinical outcomes (Li, 1998; Nathan, 1997; Li, 2001...

Genetically altering the expression of neutral trehalase gene affects conidiospore thermotolerance of the entomopathogenic fungus Metarhizium acridum

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Peng Guoxiong

2011-02-01

Full Text Available Abstract Background The entomopathogenic fungus Metarhizium acridum has been used as an important biocontrol agent instead of insecticides for controlling crop pests throughout the world. However, its virulence varies with environmental factors, especially temperature. Neutral trehalase (Ntl hydrolyzes trehalose, which plays a role in environmental stress response in many organisms, including M. acridum. Demonstration of a relationship between Ntl and thermotolerance or virulence may offer a new strategy for enhancing conidiospore thermotolerance of entomopathogenic fungi through genetic engineering. Results We selected four Ntl over-expression and four Ntl RNA interference (RNAi transformations in which Ntl expression is different. Compared to the wild-type, Ntl mRNA expression was reduced to 35-66% in the RNAi mutants and increased by 2.5-3.5-fold in the over-expression mutants. The RNAi conidiospores exhibited less trehalase activity, accumulated more trehalose, and were much more tolerant of heat stress than the wild-type. The opposite effects were found in conidiospores of over-expression mutants compared to RNAi mutants. Furthermore, virulence was not altered in the two types of mutants compared to the wild type. Conclusions Ntl controlled trehalose accumulation in M. acridum by degrading trehalose, and thus affected conidiospore thermotolerance. These results offer a new strategy for enhancing conidiospore thermotolerance of entomopathogenic fungi without affecting virulence.

A pathological study on overexpression of c-fos and Rb proteins in human radiation skin ulcer

International Nuclear Information System (INIS)

Du Yuejiao; Wang Dewen; Gao Yabing

1996-01-01

We performed an immunohistochemical study on human radiation skin ulcer by using antibodies against c-fos and Rb proteins and antigen-repairing method with a microwave oven. We found that the positive rates of overexpression of c-fos and Rb proteins were 84.0% and 100%, respectively. The overexpression of c-fos protein was mainly observed in cell nuclei of squamous epithelial cells, fibroblasts, endothelial cells, and leiomyocytes in media and fibrocytes in adventitia of arterioles. The location of the Rb protein overexpression was mostly similar to that of c-fos protein. The overexpression of c-fos and Rb proteins may be related to cancer transformation and poor healing of radiation-induced skin ulcers

MMP20 Overexpression Disrupts Molar Ameloblast Polarity and Migration.

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Shin, M; Chavez, M B; Ikeda, A; Foster, B L; Bartlett, J D

2018-02-01

Ameloblasts responsible for enamel formation express matrix metalloproteinase 20 (MMP20), an enzyme that cleaves enamel matrix proteins, including amelogenin (AMELX) and ameloblastin (AMBN). Previously, we showed that continuously erupting incisors from transgenic mice overexpressing active MMP20 had a massive cell infiltrate present within their enamel space, leading to enamel mineralization defects. However, effects of MMP20 overexpression on mouse molars were not analyzed, although these teeth more accurately represent human odontogenesis. Therefore, MMP20-overexpressing mice ( Mmp20 +/+ Tg + ) were assessed by multiscale analyses, combining several approaches from high-resolution micro-computed tomography to enamel organ immunoblots. During the secretory stage at postnatal day 6 (P6), Mmp20 +/+ Tg + mice had a discontinuous ameloblast layer and, unlike incisors, molar P12 maturation stage ameloblasts abnormally migrated away from the enamel layer into the stratum intermedium/stellate reticulum. TOPflash assays performed in vitro demonstrated that MMP20 expression promoted β-catenin nuclear localization and that MMP20 expression promoted invasion through Matrigel-coated filters. However, for both assays, significant differences were eliminated in the presence of the β-catenin inhibitor ICG-001. This suggests that MMP20 activity promotes cell migration via the Wnt pathway. In vivo, the unique molar migration of amelogenin-expressing ameloblasts was associated with abnormal deposition of ectopic calcified nodules surrounding the adherent enamel layer. Enamel content was assessed just prior to eruption at P15. Compared to wild-type, Mmp20 +/+ Tg + molars exhibited significant reductions in enamel thickness (70%), volume (60%), and mineral density (40%), and MMP20 overexpression resulted in premature cleavage of AMBN, which likely contributed to the severe defects in enamel mineralization. In addition, Mmp20 +/+ Tg + mouse molar enamel organs had increased levels

Mechanisms of MRP over-expression in four human lung-cancer cell lines and analysis of the MRP amplicon

NARCIS (Netherlands)

Eijdems, E. W.; de Haas, M.; Coco-Martin, J. M.; Ottenheim, C. P.; Zaman, G. J.; Dauwerse, H. G.; Breuning, M. H.; Twentyman, P. R.; Borst, P.; Baas, F.

1995-01-01

Some multidrug resistant cell lines over-express the gene encoding the multidrug-resistance-associated protein (MRP). In all cell lines reported thus far, over-expression is associated with gene amplification. We have studied the predominant mechanisms of MRP over-expression in 4 human lung-cancer

MECHANISMS OF MRP OVER-EXPRESSION IN 4 HUMAN LUNG-CANCER CELL-LINES AND ANALYSIS OF THE MRP AMPLICON

NARCIS (Netherlands)

EIJDEMS, EWHM; DEHAAS, M; COCOMARTIN, JM; OTTENHEIM, CPE; ZAMAN, GJR; DAUWERSE, HG; BREUNING, MH; TWENTYMAN, PR; BORST, P; BAAS, F

1995-01-01

Some multidrug resistant cell lines over-express the gene encoding the multidrug-resistance-associated protein (MRP). In all cell lines reported thus far, over-expression is associated with gene amplification. We have studied the predominant mechanisms of MRP over-expression in 4 human lung-cancer

Long-lasting pathological consequences of overexpression-induced α-synuclein spreading in the rat brain.

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Rusconi, Raffaella; Ulusoy, Ayse; Aboutalebi, Helia; Di Monte, Donato A

2018-04-01

Increased expression of α-synuclein can initiate its long-distance brain transfer, representing a potential mechanism for pathology spreading in age-related synucleinopathies, such as Parkinson's disease. In this study, the effects of overexpression-induced α-synuclein transfer were assessed over a 1-year period after injection of viral vectors carrying human α-synuclein DNA into the rat vagus nerve. This treatment causes targeted overexpression within neurons in the dorsal medulla oblongata and subsequent diffusion of the exogenous protein toward more rostral brain regions. Protein advancement and accumulation in pontine, midbrain, and forebrain areas were contingent upon continuous overexpression, because death of transduced medullary neurons resulted in cessation of spreading. Lack of sustained spreading did not prevent the development of long-lasting pathological changes. Particularly remarkable were findings in the locus coeruleus, a pontine nucleus with direct connections to the dorsal medulla oblongata and greatly affected by overexpression-induced transfer in this model. Data revealed progressive degeneration of catecholaminergic neurons that proceeded long beyond the time of spreading cessation. Neuronal pathology in the locus coeruleus was accompanied by pronounced microglial activation and, at later times, astrocytosis. Interestingly, microglial activation was also featured in another region reached by α-synuclein transfer, the central amygdala, even in the absence of frank neurodegeneration. Thus, overexpression-induced spreading, even if temporary, causes long-lasting pathological consequences in brain regions distant from the site of overexpression but anatomically connected to it. Neurodegeneration may be a consequence of severe protein burden, whereas even a milder α-synuclein accumulation in tissues affected by protein transfer could induce sustained microglial activation. © 2018 The Authors. Aging Cell published by the Anatomical Society and

The ontogenetic development of neurons containing calcium-binding proteins in the septum of the guinea pig: Late onset of parvalbumin immunoreactivity versus calbindin and calretinin.

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Hermanowicz-Sobieraj, Beata; Robak, Anna

2017-01-01

The study describes the immunoreactivity of calbindin (CB), calretinin (CR) and parvalbumin (PV), their distribution pattern and the co-distribution of CB and CR as well as CB and PV in the septum of the guinea pig during development. Immunohistochemistry was conducted on embryonic (E40, E50, E60), newborn (P0) and postnatal (P5, P10, P20, P40, P100) guinea pig brains. The presence of both CB and CR was detected at E40, while PV began to be observed at E60. Immunoreactivity for CB was constant throughout ontogeny. In contrast to CR immunoreactivity, PV immunoreactivity was higher in the postnatal stages than in the prenatal and newborn stages. Double immunostaining showed that CB co-localized with CR from E40 onwards, while with PV from P5 onwards, suggesting that CB co-operates with these proteins in the guinea pig septum during different periods of ontogeny. Our results also indicate that among the studied CaBPs, CB exhibited the highest immunoreactivity during both embryonic and postnatal development. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of overexpressed CFA/I fimbriae in bacterial swimming.

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Cao, Ling; Suo, Zhiyong; Lim, Timothy; Jun, Sangmu; Deliorman, Muhammedin; Riccardi, Carol; Kellerman, Laura; Avci, Recep; Yang, Xinghong

2012-06-01

Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 CFA/I fimbriae on bacterial swimming motility.

Enhanced production of GDP-L-fucose by overexpression of NADPH regenerator in recombinant Escherichia coli.

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Lee, Won-Heong; Chin, Young-Wook; Han, Nam Soo; Kim, Myoung-Dong; Seo, Jin-Ho

2011-08-01

Biosynthesis of guanosine 5'-diphosphate-L-fucose (GDP-L-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP(+)-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-L-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-L-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-L-fucose production. However, GDP-L-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-L-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-L-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-L-fucose concentration of 235.2 ± 3.3 mg l(-1), corresponding to a 21% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-L-fucose production in recombinant E. coli.

Improvement of daptomycin yield by overexpression of the ...

African Journals Online (AJOL)

The effects of the accessory genes flanking the non-ribosomal peptide synthetase (NRPS) genes on daptomycin production were investigated by overexpression under the control of ermE* promoter via the integrative Escherichia coli–Streptomyces vector pIB139. The yield of daptomycin was promoted significantly when ...

Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells

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Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun; Choi, Yun Jin; Kim, Yong Sang [Center for Stem Cell and Arthritis Research, Department of Orthopedic Surgery, Yonsei Sarang Hospital, Seoul (Korea, Republic of); Ko, Kinarm [Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701 (Korea, Republic of); Koh, Yong-Gon, E-mail: yonseranglab@daum.net [Center for Stem Cell and Arthritis Research, Department of Orthopedic Surgery, Yonsei Sarang Hospital, Seoul (Korea, Republic of)

2014-08-08

Highlights: • Continuous TGF-β1 overexpression in hSD-MSCs did not influence their phenotypes. • Retroviral-mediated transduction of TGFB1 in hSD-MSCs enhances cell proliferation. • TGF-β1 overexpression did not effect to adipo- or osteogenic potential of hSD-MSCs. • TGF-β1 overexpression in hSD-MSCs could stimulate and accelerate chondrogenesis. - Abstract: Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuous TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.

Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells

International Nuclear Information System (INIS)

Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun; Choi, Yun Jin; Kim, Yong Sang; Ko, Kinarm; Koh, Yong-Gon

2014-01-01

Highlights: • Continuous TGF-β1 overexpression in hSD-MSCs did not influence their phenotypes. • Retroviral-mediated transduction of TGFB1 in hSD-MSCs enhances cell proliferation. • TGF-β1 overexpression did not effect to adipo- or osteogenic potential of hSD-MSCs. • TGF-β1 overexpression in hSD-MSCs could stimulate and accelerate chondrogenesis. - Abstract: Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuous TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs

Cell division cycle 20 overexpression predicts poor prognosis for patients with lung adenocarcinoma.

Science.gov (United States)

Shi, Run; Sun, Qi; Sun, Jing; Wang, Xin; Xia, Wenjie; Dong, Gaochao; Wang, Anpeng; Jiang, Feng; Xu, Lin

2017-03-01

The cell division cycle 20, a key component of spindle assembly checkpoint, is an essential activator of the anaphase-promoting complex. Aberrant expression of cell division cycle 20 has been detected in various human cancers. However, its clinical significance has never been deeply investigated in non-small-cell lung cancer. By analyzing The Cancer Genome Atlas database and using some certain online databases, we validated overexpression of cell division cycle 20 in both messenger RNA and protein levels, explored its clinical significance, and evaluated the prognostic role of cell division cycle 20 in non-small-cell lung cancer. Cell division cycle 20 expression was significantly correlated with sex (p = 0.003), histological classification (p overexpression of cell division cycle 20 was significantly associated with bigger primary tumor size (p = 0.0023), higher MKI67 level (r = 0.7618, p Overexpression of cell division cycle 20 is associated with poor prognosis in lung adenocarcinoma patients, and its overexpression can also be used to identify high-risk groups. In conclusion, cell division cycle 20 might serve as a potential biomarker for lung adenocarcinoma patients.

Electrical remodeling of cardiac myocytes from mice with heart failure due to the overexpression of tumor necrosis factor-alpha.

Science.gov (United States)

Petkova-Kirova, Polina S; Gursoy, Erdal; Mehdi, Haider; McTiernan, Charles F; London, Barry; Salama, Guy

2006-05-01

Mice that overexpress the inflammatory cytokine tumor necrosis factor-alpha in the heart (TNF mice) develop heart failure characterized by atrial and ventricular dilatation, decreased ejection fraction, atrial and ventricular arrhythmias, and increased mortality (males > females). Abnormalities in Ca2+ handling, prolonged action potential duration (APD), calcium alternans, and reentrant atrial and ventricular arrhythmias were previously observed with the use of optical mapping of perfused hearts from TNF mice. We therefore tested whether altered voltage-gated outward K+ and/or inward Ca2+ currents contribute to the altered action potential characteristics and the increased vulnerability to arrhythmias. Whole cell voltage-clamp recordings of K+ currents from left ventricular myocytes of TNF mice revealed an approximately 50% decrease in the rapidly activating, rapidly inactivating transient outward K+ current Ito and in the rapidly activating, slowly inactivating delayed rectifier current IK,slow1, an approximately 25% decrease in the rapidly activating, slowly inactivating delayed rectifier current IK,slow2, and no significant change in the steady-state current Iss compared with controls. Peak amplitudes and inactivation kinetics of the L-type Ca2+ current ICa,L were not altered. Western blot analyses revealed a reduction in the proteins underlying Kv4.2, Kv4.3, and Kv1.5. Thus decreased K+ channel expression is largely responsible for the prolonged APD in the TNF mice and may, along with abnormalities in Ca2+ handling, contribute to arrhythmias.

Cryptochrome 1 regulates growth and development in Brassica through alteration in the expression of genes involved in light, phytohormone and stress signalling.

Science.gov (United States)

Sharma, Pooja; Chatterjee, Mithu; Burman, Naini; Khurana, Jitendra P

2014-04-01

The blue light photoreceptors cryptochromes are ubiquitous in higher plants and are vital for regulating plant growth and development. In spite of being involved in controlling agronomically important traits like plant height and flowering time, cryptochromes have not been extensively characterized from agriculturally important crops. Here we show that overexpression of CRY1 from Brassica napus (BnCRY1), an oilseed crop, results in short-statured Brassica transgenics, likely to be less prone to wind and water lodging. The overexpression of BnCRY1 accentuates the inhibition of cell elongation in hypocotyls of transgenic seedlings. The analysis of hypocotyl growth inhibition and anthocyanin accumulation responses in BnCRY1 overexpressors substantiates that regulation of seedling photomorphogenesis by cry1 is dependent on light intensity. This study highlights that the photoactivated cry1 acts through coordinated induction and suppression of specific downstream genes involved in phytohormone synthesis or signalling, and those involved in cell wall modification, during de-etiolation of Brassica seedlings. The microarray-based transcriptome profiling also suggests that the overexpression of BnCRY1 alters abiotic/biotic stress signalling pathways; the transgenic seedlings were apparently oversensitive to abscisic acid (ABA) and mannitol. © 2013 John Wiley & Sons Ltd.

[Role of autophagy in TXNIP overexpression-induced apoptosis of INS-1 islet cells].

Science.gov (United States)

Wang, Jing; Wang, Jin; Wang, Juan-Juan; Zhang, Wei-Fang; Jiao, Xiang-Ying

2017-08-25

Thioredoxin (Trx) interacting protein (TXNIP) is a Trx-binding protein that inhibits the antioxidative function of Trx and is highly expressed in the serum and tissue samples from diabetes patients. This study was to explore whether TXNIP overexpression could cause INS-1 cell autophagy under normal glucose and lipid concentrations, and to analyze the role of autophagy in the apoptosis of INS-1 cells. The INS-1 cells cultured under normal conditions were divided into three groups: normal control, empty adenovirus vector (Ad-eGFP) and TXNIP overexpression (Ad-TXNIP-eGFP) groups. Forty-eight hours after transfection, the expression levels of TXNIP mRNA and protein were measured. Western blot was used to examine the protein expression levels of Beclin-1 and P62, as well as LC3-II/LC3-I ratio, which are associated with autophagy. IF/ICC was used to measure the autophagosome. In addition, the cleaved caspase-3/caspase-3 ratio, the apoptosis marker, was also measured, and the apoptotic rates were detected by flow cytometry (FCM). The results showed that the TXNIP mRNA and protein levels were significantly up-regulated in Ad-TXNIP-eGFP group, suggesting that TXNIP overexpression model was successfully established. In Ad-TXNIP-eGFP group, the protein levels of Beclin-1 and LC3-II/LC3-I ratio were increased, while the protein expression of P62 was decreased, compared with those in Ad-eGFP group. Red fluorescent intensity, representing autophagy level, was higher in Ad-TXNIP-eGFP group than that in Ad-eGFP group. These results suggested that TXNIP overexpression can significantly promote INS-1 cell autophagy. Meanwhile, cleaved caspase 3/caspase 3 ratio and the number of apoptotic cells were significantly increased in Ad-TXNIP-eGFP group. The inhibitor of autophagy, 3-MA, reduced TXNIP overexpression-induced apoptosis in INS-1 cells. Taken together, our data demonstrate that autophagy appears to be an important pathway in TXNIP overexpression-induced apoptosis in INS-1 cells.

Overexpression of galectin-7 in mouse epidermis leads to loss of cell junctions and defective skin repair.

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Gaëlle Gendronneau

Full Text Available The proteins of the galectin family are implicated in many cellular processes, including cell interactions, polarity, intracellular trafficking, and signal transduction. In human and mouse, galectin-7 is almost exclusively expressed in stratified epithelia, notably in the epidermis. Galectin-7 expression is also altered in several human tumors of epithelial origin. This study aimed at dissecting the consequences of galectin-7 overexpression on epidermis structure and functions in vivo.We established transgenic mice specifically overexpressing galectin-7 in the basal epidermal keratinocytes and analyzed the consequences on untreated skin and after UVB irradiation or mechanical injury.The intercellular cohesion of the epidermis is impaired in transgenic animals, with gaps developing between adjacent keratinocytes, associated with loss of adherens junctions. The epidermal architecture is aberrant with perturbations in the multilayered cellular organisation of the tissue, and structural defects in the basement membrane. These transgenic animals displayed a reduced re-epithelialisation potential following superficial wound, due to a defective collective migration of keratinocytes. Finally, a single mild dose of UVB induced an abnormal apoptotic response in the transgenic epidermis.These results indicate that an excess of galectin-7 leads to a destabilisation of adherens junctions associated with defects in epidermal repair. As this phenotype shares similarities with that of galectin-7 null mutant mice, we conclude that a critical level of this protein is required for maintaining proper epidermal homeostasis. This study brings new insight into the mode of action of galectins in normal and pathological situations.

Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis1[OPEN

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Zhang, Chunyu; Zhang, Wei; Ren, Guodong; Li, Delin; Cahoon, Rebecca E.; Chen, Ming; Zhou, Yongming; Yu, Bin

2015-01-01

Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis. PMID:26048882

Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT

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Xu, Hanwen; Pirisi, Lucia; Creek, Kim E.

2015-01-01

Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial–mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. - Highlights: • Six1 expression increases during HPV16-mediated transformation. • Six1 overexpression causes differentiation resistance in HPV16-immortalized cells. • Six1 overexpression in HPV16-immortalized keratinocytes activates MAPK. • Activation of MAPK promotes EMT and differentiation resistance. • Six1 overexpression reduces Smad-dependent TGF-β signaling

Striatal dopamine transmission is subtly modified in human A53Tα-synuclein overexpressing mice.

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Nicola J Platt

Full Text Available Mutations in, or elevated dosage of, SNCA, the gene for α-synuclein (α-syn, cause familial Parkinson's disease (PD. Mouse lines overexpressing the mutant human A53Tα-syn may represent a model of early PD. They display progressive motor deficits, abnormal cellular accumulation of α-syn, and deficits in dopamine-dependent corticostriatal plasticity, which, in the absence of overt nigrostriatal degeneration, suggest there are age-related deficits in striatal dopamine (DA signalling. In addition A53Tα-syn overexpression in cultured rodent neurons has been reported to inhibit transmitter release. Therefore here we have characterized for the first time DA release in the striatum of mice overexpressing human A53Tα-syn, and explored whether A53Tα-syn overexpression causes deficits in the release of DA. We used fast-scan cyclic voltammetry to detect DA release at carbon-fibre microelectrodes in acute striatal slices from two different lines of A53Tα-syn-overexpressing mice, at up to 24 months. In A53Tα-syn overexpressors, mean DA release evoked by a single stimulus pulse was not different from wild-types, in either dorsal striatum or nucleus accumbens. However the frequency responsiveness of DA release was slightly modified in A53Tα-syn overexpressors, and in particular showed slight deficiency when the confounding effects of striatal ACh acting at presynaptic nicotinic receptors (nAChRs were antagonized. The re-release of DA was unmodified after single-pulse stimuli, but after prolonged stimulation trains, A53Tα-syn overexpressors showed enhanced recovery of DA release at old age, in keeping with elevated striatal DA content. In summary, A53Tα-syn overexpression in mice causes subtle changes in the regulation of DA release in the striatum. While modest, these modifications may indicate or contribute to striatal dysfunction.

Overexpression of phosphomimic mutated OsWRKY53 leads to enhanced blast resistance in rice.

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Tetsuya Chujo

Full Text Available WRKY transcription factors and mitogen-activated protein kinase (MAPK cascades have been shown to play pivotal roles in the regulation of plant defense responses. We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus. In this study, we identified OsWRKY53 as a substrate of OsMPK3/OsMPK6, components of a fungal PAMP-responsive MAPK cascade in rice, and analyzed the effect of OsWRKY53 phosphorylation on the regulation of basal defense responses to a virulence race of rice blast fungus Magnaporthe oryzae strain Ina86-137. An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster. When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster. Transgenic rice plants overexpressing a phospho-mimic mutant of OsWRKY53 (OsWRKY53SD showed further-enhanced disease resistance to the blast fungus compared to native OsWRKY53-overexpressing rice plants, and a substantial number of defense-related genes, including pathogenesis-related protein genes, were more upregulated in the OsWRKY53SD-overexpressing plants compared to the OsWRKY53-overexpressing plants. These results strongly suggest that the OsMKK4-OsMPK3/OsMPK6 cascade regulates transactivation activity of OsWRKY53, and overexpression of the phospho-mimic mutant of OsWRKY53 results in a major change to the rice transcriptome at steady state that leads to activation of a defense response against the blast fungus in rice plants.

Overexpression of Phosphomimic Mutated OsWRKY53 Leads to Enhanced Blast Resistance in Rice

Science.gov (United States)

Ogawa, Satoshi; Masuda, Yuka; Shimizu, Takafumi; Kishi-Kaboshi, Mitsuko; Takahashi, Akira; Nishizawa, Yoko; Minami, Eiichi; Nojiri, Hideaki; Yamane, Hisakazu; Okada, Kazunori

2014-01-01

WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses. We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus. In this study, we identified OsWRKY53 as a substrate of OsMPK3/OsMPK6, components of a fungal PAMP-responsive MAPK cascade in rice, and analyzed the effect of OsWRKY53 phosphorylation on the regulation of basal defense responses to a virulence race of rice blast fungus Magnaporthe oryzae strain Ina86-137. An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster). When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster. Transgenic rice plants overexpressing a phospho-mimic mutant of OsWRKY53 (OsWRKY53SD) showed further-enhanced disease resistance to the blast fungus compared to native OsWRKY53-overexpressing rice plants, and a substantial number of defense-related genes, including pathogenesis-related protein genes, were more upregulated in the OsWRKY53SD-overexpressing plants compared to the OsWRKY53-overexpressing plants. These results strongly suggest that the OsMKK4-OsMPK3/OsMPK6 cascade regulates transactivation activity of OsWRKY53, and overexpression of the phospho-mimic mutant of OsWRKY53 results in a major change to the rice transcriptome at steady state that leads to activation of a defense response against the blast fungus in rice plants. PMID:24892523

Overexpression of Hypoxia-Inducible Factor-1α Exacerbates Endothelial Barrier Dysfunction Induced by Hypoxia

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Pei Wang

2013-09-01

Full Text Available Background/Aims: The mechanisms involved in endothelial barrier dysfunction induced by hypoxia are incompletely understood. There is debate about the role of hypoxia-inducible factor-1α (HIF-1α in endothelial barrier disruption. The aim of this study was to investigate the effect of genetic overexpression of HIF-1α on barrier function and the underlying mechanisms in hypoxic endothelial cells. Methods: The plasmid pcDNA3.1/V5-His-HIF-1α was stably transfected into human endothelial cells. The cells were exposed to normoxia or hypoxia. The mRNA and protein expressions of HIF-1α were detected by RT-PCR and Western blot respectively. The barrier function was assessed by measuring the transendothelial electrical resistance (TER. The Western blot analysis was used to determine the protein expression of glucose transporter-1 (GLUT-1, zonular occludens-1 (ZO-1, occludin, and myosin light chain kinase (MLCK in endothelial cells. The mRNA expression of proinflammatory cytokines was detected by qRT-PCR. Results: Genetic overexpression of HIF-1α significantly increased the mRNA and protein expression of HIF-1α in endothelial cells. The overexpression of HIF-1α enhanced the hypoxia-induced increase of HIF-1α and GLUT-1 protein expression. HIF-1α overexpression not only exacerbated hypoxia-induced endothelial barrier dysfunction but also augmented hypoxia-induced up-regulation of MLCK protein expression. HIF-1α overexpression also enhanced IL-1β, IL-6 and TNF-α mRNA expression. Conclusion: We provide evidence that genetic overexpression of HIF-1α aggravates the hypoxia-induced endothelial barrier dysfunction via enhancing the up-regulation of MLCK protein expression caused by hypoxia, suggesting a potential role for HIF-1α in the pathogenesis of endothelial barrier dysfunction in hypoxia.

LncRNA CCAT2 promotes tumorigenesis by over-expressed Pokemon in non-small cell lung cancer.

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Zhao, Zhihong; Wang, Ju; Wang, Shengfa; Chang, Hao; Zhang, Tiewa; Qu, Junfeng

2017-03-01

Non-small cell lung cancer (NSCLC) remains one of the most important death-related diseases, with poor effective diagnosis and less therapeutic biomarkers. LncRNA colon cancer-associated transcript 2 (CCAT2) was identified as an oncogenic lncRNA and over-expressed in many tumor cells. The aims of this study were to detect the correlation between CCAT2 and its regulatory genes and then explore the potential mechanism between them in NSCLC. In this study, qRT-PCR was used to detect CCAT2, Pokemon and p21 expression. Western-blot was used to detect protein levels of Pokemon and p21. CCK-8 assay and Transwell chambers were used to assess cell viability and invasion. CCAT2 and Pokemon were over-expressed in NSCLC tissue and cells. In NSCLC cells, CCAT2 knockdown significantly decreased cell viability and invasion as well as Pokemon expression, but increased the expression of p21; then CCAT2 overexpression revealed an opposite result. In addition, over-expressed Pokemon reversed the results that induced by si-CCAT2, while down-regulation of Pokemon significantly reversed the results that induced by CCAT2 overexpression. The results indicated that CCAT2 promotes tumorigenesis by over-expression of Pokemon, and the potential mechanism might relate to the Pokemon related gene p21. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Cyclin-dependent kinase inhibitor 3 is overexpressed in hepatocellular carcinoma and promotes tumor cell proliferation

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Xing, Chunyang; Xie, Haiyang; Zhou, Lin; Zhou, Wuhua; Zhang, Wu; Ding, Songming; Wei, Bajin; Yu, Xiaobo; Su, Rong; Zheng, Shusen

2012-01-01

Highlights: ► CDKN3 is commonly overexpressed in HCC and is associated with poor clinical outcome. ► Overexpression of CDKN3 could stimulate the proliferation of HCC cells by promoting G1/S transition. ► CDKN3 could inhibit the expression of p21 in HCC cells. ► Overexpression of CDKN3 has no effect on apoptosis and invasion of HCC cells. ► We identified 61 genes co-expressed with CDKN3, and BIRC5 was located at the center of the co-expression network. -- Abstract: Cyclin-dependent kinase inhibitor 3 (CDKN3) belongs to the protein phosphatases family and has a dual function in cell cycling. The function of this gene has been studied in several kinds of cancers, but its role in human hepatocellular carcinoma (HCC) remains to be elucidated. In this study, we found that CDKN3 was frequently overexpressed in both HCC cell lines and clinical samples, and this overexpression was correlated with poor tumor differentiation and advanced tumor stage. Functional studies showed that overexpression of CDKN3 could promote cell proliferation by stimulating G1-S transition but has no impact on cell apoptosis and invasion. Microarray-based co-expression analysis identified a total of 61 genes co-expressed with CDKN3, with most of them involved in cell proliferation, and BIRC5 was located at the center of CDKN3 co-expression network. These results suggest that CDKN3 acts as an oncogene in human hepatocellular carcinoma and antagonism of CDKN3 may be of interest for the treatment of HCC.

Functional overexpression and characterization of lipogenesis-related genes in the oleaginous yeast Yarrowia lipolytica.

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Silverman, Andrew M; Qiao, Kangjian; Xu, Peng; Stephanopoulos, Gregory

2016-04-01

Single cell oil (SCO) is an attractive energy source due to scalability, utilization of low-cost renewable feedstocks, and type of product(s) made. Engineering strains capable of producing high lipid titers and yields is crucial to the economic viability of these processes. However, lipid synthesis in cells is a complex phenomenon subject to multiple layers of regulation, making gene target identification a challenging task. In this study, we aimed to identify genes in the oleaginous yeast Yarrowia lipolytica whose overexpression enhances lipid production by this organism. To this end, we examined the effect of the overexpression of a set of 44 native genes on lipid production in Y. lipolytica, including those involved in glycerolipid synthesis, fatty acid synthesis, central carbon metabolism, NADPH generation, regulation, and metabolite transport and characterized each resulting strain's ability to produce lipids growing on both glucose and acetate as a sole carbon source. Our results suggest that a diverse subset of genes was effective at individually influencing lipid production in Y. lipolytica, sometimes in a substrate-dependent manner. The most productive strain on glucose overexpressed the diacylglycerol acyltransferase DGA2 gene, increasing lipid titer, cellular content, and yield by 236, 165, and 246 %, respectively, over our control strain. On acetate, our most productive strain overexpressed the acylglycerol-phosphate acyltransferase SLC1 gene, with a lipid titer, cellular content, and yield increase of 99, 91, and 151 %, respectively, over the control strain. Aside from genes encoding enzymes that directly catalyze the reactions of lipid synthesis, other ways by which lipogenesis was increased in these cells include overexpressing the glycerol-3-phosphate dehydrogenase (GPD1) gene to increase production of glycerol head groups and overexpressing the 6-phosphogluconolactonase (SOL3) gene from the oxidative pentose phosphate pathway to increase NADPH

Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans

DEFF Research Database (Denmark)

Schalén, Martin; Anyaogu, Diana Chinyere; Hoof, Jakob Blæsbjerg

2016-01-01

roles in the process have been identified through transcriptomics. The assignment of function to these genes has been enabled in combination with gene deletion studies. In this work, 14 genes known to play a role in protein secretion in filamentous fungi were overexpressed in Aspergillus nidulans....... The background strain was a fluorescent reporter secreting mRFP. The overall effect of the overexpressions could thus be easily monitored through fluorescence measurements, while the effects on physiology were determined in batch cultivations and surface growth studies. Results: Fourteen protein secretion...... pathway related genes were overexpressed with a tet-ON promoter in the RFP-secreting reporter strain and macromorphology, physiology and protein secretion were monitored when the secretory genes were induced. Overexpression of several of the chosen genes was shown to cause anomalies on growth, micro...

Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect

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Ghezali, Lamia; Leger, David Yannick; Limami, Youness; Cook-Moreau, Jeanne; Beneytout, Jean-Louis; Liagre, Bertrand

2013-01-01

Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effect on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines. - Highlights: ► Cyclopamine alone but not jervine induces apoptosis in human erythroleukemia cells. ► Cyclopamine and jervine induce COX-2 overexpression. ► COX-2 overexpression is implicated in resistance to cyclopamine-induced apoptosis. ► Apoptotic potential of jervine is restrained by NF-κB pathway activation. ► PKC is involved in cyclopamine-induced apoptosis and COX-2 overexpression

Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect

Energy Technology Data Exchange (ETDEWEB)

Ghezali, Lamia; Leger, David Yannick; Limami, Youness [Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France); Cook-Moreau, Jeanne [Université de Limoges, FR 3503 GEIST, UMR CNRS 7276 “Contrôle de la réponse immune B et lymphoproliférations”, Faculté de Médecine, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France); Beneytout, Jean-Louis [Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France); Liagre, Bertrand, E-mail: bertrand.liagre@unilim.fr [Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France)

2013-04-15

Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effect on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines. - Highlights: ► Cyclopamine alone but not jervine induces apoptosis in human erythroleukemia cells. ► Cyclopamine and jervine induce COX-2 overexpression. ► COX-2 overexpression is implicated in resistance to cyclopamine-induced apoptosis. ► Apoptotic potential of jervine is restrained by NF-κB pathway activation. ► PKC is involved in cyclopamine-induced apoptosis and COX-2 overexpression.

Atrial SERCA2a Overexpression Has No Affect on Cardiac Alternans but Promotes Arrhythmogenic SR Ca2+ Triggers.

Science.gov (United States)

Nassal, Michelle M J; Wan, Xiaoping; Laurita, Kenneth R; Cutler, Michael J

2015-01-01

Atrial fibrillation (AF) is the most common arrhythmia in humans, yet; treatment has remained sub-optimal due to poor understanding of the underlying mechanisms. Cardiac alternans precede AF episodes, suggesting an important arrhythmia substrate. Recently, we demonstrated ventricular SERCA2a overexpression suppresses cardiac alternans and arrhythmias. Therefore, we hypothesized that atrial SERCA2a overexpression will decrease cardiac alternans and arrhythmias. Adult rat isolated atrial myocytes where divided into three treatment groups 1) Control, 2) SERCA2a overexpression (Ad.SERCA2a) and 3) SERCA2a inhibition (Thapsigargin, 1μm). Intracellular Ca2+ was measured using Indo-1AM and Ca2+ alternans (Ca-ALT) was induced with a standard ramp pacing protocol. As predicted, SR Ca2+ reuptake was enhanced with SERCA2a overexpression (poverexpression or inhibition when compared to controls (p = 0.73). In contrast, SERCA2a overexpression resulted in increased premature SR Ca2+ (SCR) release compared to control myocytes (28% and 0%, p overexpression in atrial myocytes can increase SCR, which may be arrhythmogenic.

Overexpression of human and fly frataxins in Drosophila provokes deleterious effects at biochemical, physiological and developmental levels.

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Juan A Navarro

Full Text Available BACKGROUND: Friedreich's ataxia (FA, the most frequent form of inherited ataxias in the Caucasian population, is caused by a reduced expression of frataxin, a highly conserved protein. Model organisms have contributed greatly in the efforts to decipher the function of frataxin; however, the precise function of this protein remains elusive. Overexpression studies are a useful approach to investigate the mechanistic actions of frataxin; however, the existing literature reports contradictory results. To further investigate the effect of frataxin overexpression, we analyzed the consequences of overexpressing human (FXN and fly (FH frataxins in Drosophila. METHODOLOGY/PRINCIPAL FINDINGS: We obtained transgenic flies that overexpressed human or fly frataxins in a general pattern and in different tissues using the UAS-GAL4 system. For both frataxins, we observed deleterious effects at the biochemical, histological and behavioral levels. Oxidative stress is a relevant factor in the frataxin overexpression phenotypes. Systemic frataxin overexpression reduces Drosophila viability and impairs the normal embryonic development of muscle and the peripheral nervous system. A reduction in the level of aconitase activity and a decrease in the level of NDUF3 were also observed in the transgenic flies that overexpressed frataxin. Frataxin overexpression in the nervous system reduces life span, impairs locomotor ability and causes brain degeneration. Frataxin aggregation and a misfolding of this protein have been shown not to be the mechanism that is responsible for the phenotypes that have been observed. Nevertheless, the expression of human frataxin rescues the aconitase activity in the fh knockdown mutant. CONCLUSION/SIGNIFICANCE: Our results provide in vivo evidence of a functional equivalence for human and fly frataxins and indicate that the control of frataxin expression is important for treatments that aim to increase frataxin levels.

[Overexpressed miRNA-134b inhibits proliferation and invasion of CD133+ U87 glioma stem cells].

Science.gov (United States)

Liu, Yifeng; Zhang, Baochao; Wen, Changming; Wen, Gongling; Zhou, Guoping; Zhang, Jingwei; He, Haifa; Wang, Ning; Li, Wei

2017-05-01

Objective To investigate the role of microRNA-134b (miR-134b) in the tumorigenesis of glioma stem cells (GSCs) and the possible molecular mechanism. Methods Real-time quantitative PCR (qRT-PCR) was used to evalate the expression of miR-134b in CD133 + and CD133 - U87 GSCs. A lentiviral vector overexpressing miR-134b in U87 GSCs was constructed, and the effect of miR-134b overexpression on matrix metalloproteinase-2 (MMP-2), MMP-9 and MMP-12 expressions at both mRNA and protein levels were detected by qRT-PCR and Western blotting, respectively. Transwell TM assay was performed to determine the effect of miR-134b overexpression on GSCs invasion ability. Tumor xenograft models in nude mice were established to evaluate the effect of miR-134b overexpression on tumorgenesis in vivo. Results The qRT-PCR showed that, compared with CD133 - cells, miR-134b was significantly down-regulated in CD133 + cells. Cell line over-expressing miR-134b was successfully established, and miR-134b was up-regulated significantly compared with empty vector control. Overexpression of miR-134b remarkably inhibited the invasion of U87 GSCs and the expression of MMP-12. However, overexpression of miR-134b did not affect MMP-2 and MMP-9 expressions. miR-134b also suppressed U87 GSCs xenograft growth in vivo. Tumor volume in tumor xenograft model group was significantly lower than that in control group, and tumor weight decreased by 42% in the former group. Conclusion Overexpression of miR-134b inhibits the growth and invasion of CD133 + GSCs.

The inhibition of superoxide production in EL4 lymphoma cells overexpressing growth hormone.

Science.gov (United States)

Arnold, Robyn E; Weigent, Douglas A

2003-05-01

A substantial body of research exists to support the production of growth hormone by cells of the immune system. However, the function and mechanism of action of lymphocyte-derived growth hormone remain largely unelucidated. Since, it has been found that exogenous growth hormone (GH) primes neutrophils for the production of reactive oxygen intermediates (ROI) and in particular superoxide (O2-), we investigated the role of GH on the production of O2- in T cells. Furthermore, we examined whether endogenous and exogenous GH act similarly. Our studies show that overexpression of GH in EL4, a T-cell lymphoma cell line, results in a decrease in the production of O2- compared to control cells, as detected using the fluorescent dye, dihydroethidium. O2- production in control cells was not affected by treatment with inhibitors of xanthine oxidase or a non-specific NADPH-oxidase inhibitor. However, treatment with diallyl sulfide, an inhibitor of cytochrome P450 2E1 mimicked the reduction in O2- production seen in cells overexpressing GH. Although no significant change could be detected in CYP2E1 protein levels, CYP2E1 activity was found to be greater in control EL4 than in cells overexpressing GH. Both the decrease in O2- production and the lower CYP2E1 activity in GH overexpressing cells could be abrogated by treatment with N(G)-monomethyl-L-arginine, an inhibitor of nitric oxide synthase. The overexpression of GH protects cells from apoptosis induced by isoniazid, a CYP2E1 inducer, suggesting a role for nitric oxide as a mediator in the regulation of xenobiotic metabolism and apoptosis-protection by lymphocyte GH.

Oncogene alterations in carcinomas of the uterine cervix: overexpression of the epidermal growth factor receptor is associated with poor prognosis

NARCIS (Netherlands)

Kersemaekers, A. M.; Fleuren, G. J.; Kenter, G. G.; van den Broek, L. J.; Uljee, S. M.; Hermans, J.; van de Vijver, M. J.

1999-01-01

The involvement of human papillomavirus (HPV) in the development of carcinomas of the uterine cervix has been firmly established. However, other genetic alterations also play an important role in the pathogenesis of cervical cancer. Therefore, we have investigated the role of several (onco)genes in

Oncolytic adenovirus targeting cyclin E overexpression repressed tumor growth in syngeneic immunocompetent mice

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Cheng, Pei-Hsin; Rao, Xiao-Mei; Wechman, Stephen L.; Li, Xiao-Feng; McMasters, Kelly M.; Zhou, Heshan Sam

2015-01-01

Clinical trials have indicated that preclinical results obtained with human tumor xenografts in mouse models may overstate the potential of adenovirus (Ad)-mediated oncolytic therapies. We have previously demonstrated that the replication of human Ads depends on cyclin E dysregulation or overexpression in cancer cells. ED-1 cell derived from mouse lung adenocarcinomas triggered by transgenic overexpression of human cyclin E may be applied to investigate the antitumor efficacy of oncolytic Ads. Ad-cycE was used to target cyclin E overexpression in ED-1 cells and repress tumor growth in a syngeneic mouse model for investigation of oncolytic virotherapies. Murine ED-1 cells were permissive for human Ad replication and Ad-cycE repressed ED-1 tumor growth in immunocompetent FVB mice. ED-1 cells destroyed by oncolytic Ads in tumors were encircled in capsule-like structures, while cells outside the capsules were not infected and survived the treatment. Ad-cycE can target cyclin E overexpression in cancer cells and repress tumor growth in syngeneic mouse models. The capsule structures formed after Ad intratumoral injection may prevent viral particles from spreading to the entire tumor. The online version of this article (doi:10.1186/s12885-015-1731-x) contains supplementary material, which is available to authorized users

Overexpression of the DYRK1A Gene (Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A) Induces Alterations of the Serotoninergic and Dopaminergic Processing in Murine Brain Tissues.

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London, Jacqueline; Rouch, Claude; Bui, Linh Chi; Assayag, Elodie; Souchet, Benoit; Daubigney, Fabrice; Medjaoui, Hind; Luquet, Serge; Magnan, Christophe; Delabar, Jean Maurice; Dairou, Julien; Janel, Nathalie

2018-05-01

Trisomy 21 (T21) or Down syndrome (DS) is the most common genetic disorder associated with intellectual disability and affects around 5 million persons worldwide. Neuroanatomical phenotypes associated with T21 include slight reduction of brain size and weight, abnormalities in several brain areas including spines dysgenesis, dendritic morphogenesis, and early neuroanatomical characteristics of Alzheimer's disease. Monoamine neurotransmitters are involved in dendrites development, functioning of synapses, memory consolidation, and their levels measured in the cerebrospinal fluid, blood, or brain areas that are modified in individuals with T21. DYRK1A is one of the recognized key genes that could explain some of the deficits present in individuals with T21. We investigated by high-performance liquid chromatography with electrochemical detection the contents and processing of monoamines neurotransmitters in four brain areas of female and male transgenic mice for the Dyrk1a gene (mBactgDyrk1a). DYRK1A overexpression induced dramatic deficits in the serotonin contents of the four brain areas tested and major deficits in dopamine and adrenaline contents especially in the hypothalamus. These results suggest that DYRK1A overexpression might be associated with the modification of monoamines content found in individuals with T21 and reinforce the interest to target the level of DYRK1A expression as a therapeutic approach for persons with T21.

Overexpression of PGC-1α Increases Fatty Acid Oxidative Capacity of Human Skeletal Muscle Cells

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Nataša Nikolić

2012-01-01

Full Text Available We investigated the effects of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α overexpression on the oxidative capacity of human skeletal muscle cells ex vivo. PGC-1α overexpression increased the oxidation rate of palmitic acid and mRNA expression of genes regulating lipid metabolism, mitochondrial biogenesis, and function in human myotubes. Basal and insulin-stimulated deoxyglucose uptake were decreased, possibly due to upregulation of PDK4 mRNA. Expression of fast fiber-type gene marker (MHCIIa was decreased. Compared to skeletal muscle in vivo, PGC-1α overexpression increased expression of several genes, which were downregulated during the process of cell isolation and culturing. In conclusion, PGC-1α overexpression increased oxidative capacity of cultured myotubes by improving lipid metabolism, increasing expression of genes involved in regulation of mitochondrial function and biogenesis, and decreasing expression of MHCIIa. These results suggest that therapies aimed at increasing PGC-1α expression may have utility in treatment of obesity and obesity-related diseases.

Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas.

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Calin, George A; Liu, Chang-gong; Ferracin, Manuela; Hyslop, Terry; Spizzo, Riccardo; Sevignani, Cinzia; Fabbri, Muller; Cimmino, Amelia; Lee, Eun Joo; Wojcik, Sylwia E; Shimizu, Masayoshi; Tili, Esmerina; Rossi, Simona; Taccioli, Cristian; Pichiorri, Flavia; Liu, Xiuping; Zupo, Simona; Herlea, Vlad; Gramantieri, Laura; Lanza, Giovanni; Alder, Hansjuerg; Rassenti, Laura; Volinia, Stefano; Schmittgen, Thomas D; Kipps, Thomas J; Negrini, Massimo; Croce, Carlo M

2007-09-01

Noncoding RNA (ncRNA) transcripts are thought to be involved in human tumorigenesis. We report that a large fraction of genomic ultraconserved regions (UCRs) encode a particular set of ncRNAs whose expression is altered in human cancers. Genome-wide profiling revealed that UCRs have distinct signatures in human leukemias and carcinomas. UCRs are frequently located at fragile sites and genomic regions involved in cancers. We identified certain UCRs whose expression may be regulated by microRNAs abnormally expressed in human chronic lymphocytic leukemia, and we proved that the inhibition of an overexpressed UCR induces apoptosis in colon cancer cells. Our findings argue that ncRNAs and interaction between noncoding genes are involved in tumorigenesis to a greater extent than previously thought.

Synergistic Effects of NDRG2 Overexpression and Radiotherapy on Cell Death of Human Prostate LNCaP Cells.

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Alizadeh Zarei, M; Takhshid, M A; Behzad Behbahani, A; Hosseini, S Y; Okhovat, M A; Rafiee Dehbidi, Gh R; Mosleh Shirazi, M A

2017-09-01

Radiation therapy is among the most conventional cancer therapeutic modalities with effective local tumor control. However, due to the development of radio-resistance, tumor recurrence and metastasis often occur following radiation therapy. In recent years, combination of radiotherapy and gene therapy has been suggested to overcome this problem. The aim of the current study was to explore the potential synergistic effects of N-Myc Downstream-Regulated Gene 2 (NDRG2) overexpression, a newly identified candidate tumor suppressor gene, with radiotherapy against proliferation of prostate LNCaP cell line. In this study, LNCaP cells were exposed to X-ray radiation in the presence or absence of NDRG2 overexpression using plasmid PSES- pAdenoVator-PSA-NDRG2-IRES-GFP. The effects of NDRG2 overexpression, X-ray radiation or combination of both on the cell proliferation and apoptosis of LNCaP cells were then analyzed using MTT assay and flow cytometery, respectively. Results of MTT assay showed that NDRG2 overexpression and X-ray radiation had a synergistic effect against proliferation of LNCaP cells. Moreover, NDRG2 overexpression increased apoptotic effect of X-ray radiation in LNCaP cells synergistically. Our findings suggested that NDRG2 overexpression in combination with radiotherapy may be an effective therapeutic option against prostate cancer.

Aberrant overexpression of vascular endothelial growth factor in pancreatic ductal adenocarcinoma is associated with aggressive clinical behavior

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Naomi Y Jiang

2010-07-01

Full Text Available Naomi Y Jiang1, Bruce A Woda2, Liping Zhang2, Suyang Hao2, Karen A Dresser2, Di Lu21Massachusetts Institute of Technology, Worcester, MA, USA; 2Department of Pathology, University of Massachusetts Medical Center, Worcester, MA, USAAbstract: Pancreatic adenocarcinoma is a leading cause of cancer-related deaths in the United States. In this study, we studied vascular endothelial growth factor (VEGF expression in ­pancreatic adenocarcinoma by immunohistochemical staining. Clinical follow-up and survival data were analyzed. We determined that VEGF was aberrantly overexpressed in a subset of primary pancreatic adenocarcinoma. Statistically, VEGF overexpression was associated with higher stage, higher grade, and lymph node metastasis (P < 0.001, P = 0.012, and P < 0.005, respectively. Additionally, patients of this subset had a much shorter overall survival than patients without VEGF overexpression, as evidenced by Kaplan–Meier plots and the log-rank test (P = 0.001. The 5-year overall survival rate was 17% in patients with VEGF overexpression compared to 52% in patients without VEGF overexpression. The median survival was only 13 months for patients with VEGF overexpression compared to 65 months for patients without. In conclusion, VEGF is a biomarker that identifies a subset of pancreatic ductal adenocarcinoma with aggressive clinical behavior.Keywords: pancreatic adenocarcinoma, VEGF, cancer

Redox Dysregulation in the Pathophysiology of Schizophrenia and Bipolar Disorder

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Kulak, Anita; Steullet, Pascal; Cabungcal, Jan-Harry

2013-01-01

Abstract Significance: Schizophrenia (SZ) and bipolar disorder (BD) are classified as two distinct diseases. However, accumulating evidence shows that both disorders share genetic, pathological, and epidemiological characteristics. Based on genetic and functional findings, redox dysregulation due...... abnormal prefrontal levels of glutathione (GSH), the major cellular redox regulator and antioxidant. Here we review experimental data from rodent models demonstrating that permanent as well as transient GSH deficit results in behavioral, morphological, electrophysiological, and neurochemical alterations...... hypofunction, elevated glutamate levels, impairment of parvalbumin GABA interneurons, abnormal neuronal synchronization, altered dopamine neurotransmission, and deficient myelination. Critical Issues: Treatment with the GSH precursor and antioxidant N-acetylcysteine normalizes some of those deficits in mice...

Overexpression of isocitrate lyase-glyoxylate bypass influence on metabolism in Aspergillus niger

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Meijer, Susan Lisette; Otero, José Manuel; Olivares Hernandez, Roberto

2009-01-01

of the cells was investigated. Inhibition of SDH was expected to lead to succinate production, but this was not observed. There was an increase in citrate and oxalate production in the wild-type strain. Furthermore, in the strain with over-expression of icl the organic acid production shifted from fumarate...... towards malate production when malonate was added to the cultivation medium. Overall, the icl over-expression and malonate addition had a significant impact on metabolism and on organic acid production profiles. Although the expected succinate and malate formation was not observed, a distinct...

Combined gene overexpression of neuropeptide Y and its receptor Y5 in the hippocampus suppresses seizures

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Gøtzsche, Casper René; Nikitidou, Litsa; Sørensen, Andreas Toft

2012-01-01

We recently demonstrated that recombinant adeno-associated viral vector-induced hippocampal overexpression of neuropeptide Y receptor, Y2, exerts a seizure-suppressant effect in kindling and kainate-induced models of epilepsy in rats. Interestingly, additional overexpression of neuropeptide Y...

Interleukin-6 overexpression induces pulmonary hypertension.

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Steiner, M Kathryn; Syrkina, Olga L; Kolliputi, Narasaish; Mark, Eugene J; Hales, Charles A; Waxman, Aaron B

2009-01-30

Inflammatory cytokine interleukin (IL)-6 is elevated in the serum and lungs of patients with pulmonary artery hypertension (PAH). Several animal models of PAH cite the potential role of inflammatory mediators. We investigated role of IL-6 in the pathogenesis of pulmonary vascular disease. Indices of pulmonary vascular remodeling were measured in lung-specific IL-6-overexpressing transgenic mice (Tg(+)) and compared to wild-type (Tg(-)) controls in both normoxic and chronic hypoxic conditions. The Tg(+) mice exhibited elevated right ventricular systolic pressures and right ventricular hypertrophy with corresponding pulmonary vasculopathic changes, all of which were exacerbated by chronic hypoxia. IL-6 overexpression increased muscularization of the proximal arterial tree, and hypoxia enhanced this effect. It also reproduced the muscularization and proliferative arteriopathy seen in the distal arteriolar vessels of PAH patients. The latter was characterized by the formation of occlusive neointimal angioproliferative lesions that worsened with hypoxia and were composed of endothelial cells and T-lymphocytes. IL-6-induced arteriopathic changes were accompanied by activation of proangiogenic factor, vascular endothelial growth factor, the proproliferative kinase extracellular signal-regulated kinase, proproliferative transcription factors c-MYC and MAX, and the antiapoptotic proteins survivin and Bcl-2 and downregulation of the growth inhibitor transforming growth factor-beta and proapoptotic kinases JNK and p38. These findings suggest that IL-6 promotes the development and progression of pulmonary vascular remodeling and PAH through proproliferative antiapoptotic mechanisms.

Targeted Overexpression of Inducible 6-Phosphofructo-2-kinase in Adipose Tissue Increases Fat Deposition but Protects against Diet-induced Insulin Resistance and Inflammatory Responses*

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Huo, Yuqing; Guo, Xin; Li, Honggui; Xu, Hang; Halim, Vera; Zhang, Weiyu; Wang, Huan; Fan, Yang-Yi; Ong, Kuok Teong; Woo, Shih-Lung; Chapkin, Robert S.; Mashek, Douglas G.; Chen, Yanming; Dong, Hui; Lu, Fuer; Wei, Lai; Wu, Chaodong

2012-01-01

Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues. PMID:22556414

HER-2/neu and CD117 (c-kit overexpression in patients with pesticide exposure and extensive stage small cell lung carcinoma (ESSCLC

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Potti Anil

2005-06-01

Full Text Available Abstract Background The rate of detection of HER-2/neu and CD117 (c-kit overexpression in small cell lung cancer (SCLC has varied widely; between 5–35% and 21–70% respectively. Methods To evaluate the relationship between pesticide exposure and HER-2/neu and CD117 overexpression in extensive stage SCLC (ESSCLC, we identified patients with ESSCLC and assessed pesticide exposure using a predetermined questionnaire. An exposure index (hours/day × days/year × years ≥ 2400 hours was considered as 'exposed.' HER-2/neu overexpression was evaluated on archival tissue using the DAKO Hercep test, and CD117 testing was performed using immunohistochemistry (A4052 polyclonal antibody. Results 193 ESSCLC patients were identified. Pesticide exposure data could be obtained on 174 patients (84 females and 109 males with a mean age of 68.5 years. 53/174 (30.4% revealed HER-2/neu overexpression. 54/174 (31.03% specimens showed CD117 overexpression by IHC. On multivariate analysis, HER-2/neu overexpression was associated with diminished survival (p neu overexpression and 47/121 (38.8% patients without overexpression had exposure to pesticides (odds ratio: 5.38; p Conclusion Pesticide exposure affects HER-2/neu but not CD117 overexpression. Future studies are needed to determine specific pesticide(s/pesticide components that are responsible for HER-2/neu overexpression in ESSCLC, and to validate our findings in other solid tumors that overexpress HER-2/neu.

Monitoring in real time the effect of TLX overexpression on proliferation and migration of C6 cells.

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Li, G L; Fang, S H; Xu, B

2017-01-01

Orphan nuclear receptor TLX has been shown to play an essential role in regulating the self-renewal and proliferation of neural stem cells (NSCs). However, TLX overexpression in NSCs induces long-term NSC expansion and further leads to glioma initiation in mouse when combined with p53 mutations. Whether overexpression of TLX plays a role in glioma stem cell (GSC) proliferation and migration still remains largely unknown. In this study, we infected C6 cells, a special glioma cell line which is mainly composed of cancer stem cells(CSCs), with lentiviruses expressing GFP(LV-GFP) or GFP-T2A-TLX(LV-TLX) and then monitored cell proliferation and migration using the real-time analyzer system (RTCA, xCELLigence, Roche). We found that the cell index (CI) observed for the TLX overexpressing C6 cells showed a lower value than that of the LV-GFP transduced cells. And the MTT results correlated highly with the RTCA proliferation assessments. Furthermore, the expression of p21 was decreased while other downstream genes PTEN and p53 were not significantly changed in TLX overexpressing C6 cells . These findings strongly indicate that TLX overexpression has the ability to decrease the proliferating and migratory properties of C6 cells by targeting p21. Further, our results suggest that TLX overexpression may also have a similar inhibitory effect on GSC proliferation and migration.

Overexpression of HIF-1α in mesenchymal stem cells contributes to repairing hypoxic-ischemic brain damage in rats.

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Lin, Deju; Zhou, Liping; Wang, Biao; Liu, Lizhen; Cong, Li; Hu, Chuanqin; Ge, Tingting; Yu, Qin

2017-01-01

Preclinical researches on mesenchymal stem cells (MSCs) transplantation, which is used to treat hypoxic-ischemic (HI) brain damage, have received inspiring achievements. However, the insufficient migration of active cells to damaged tissues has limited their potential therapeutic effects. There are some evidences that hypoxia inducible factor-1 alpha (HIF-1α) promotes the viability and migration of the cells. Here, we aim to investigate whether overexpression of HIF-1α in MSCs could improve the viability and migration capacity of cells, and its therapeutic efficiency on HI brain damage. In the study, MSCs with HIF-1α overexpression was achieved by recombinant lentiviral vector and transplanted to the rats subsequent to HI. Our data indicated that overexpression of HIF-1α promoted the viability and migration of MSCs, HIF-1α overexpressed MSCs also had a stronger therapeutic efficiency on HI brain damaged treatment by mitigating the injury on behavioral and histological changes evoked by HI insults, accompanied with more MSCs migrating to cerebral damaged area. This study demonstrated that HIF-1α overexpression could increase the MSCs' therapeutic efficiency in HI and the promotion of the cells' directional migration to cerebral HI area by overexpression may be responsible for it, which showed that transplantation of MSCs with HIF-1α overexpression is an attractive therapeutic option to treat HI-induced brain injury in the future. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Matrix-Dependent Regulation of AKT in Hepsin-Overexpressing PC3 Prostate Cancer Cells

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Stephanie M Wittig-Blaich

2011-07-01

Full Text Available The serine-protease hepsin is one of the most prominently overexpressed genes in human prostate carcinoma. Forced expression of the enzyme in mice prostates is associated with matrix degradation, invasive growth, and prostate cancer progression. Conversely, hepsin overexpression in metastatic prostate cancer cell lines was reported to induce cell cycle arrest and reduction of invasive growth in vitro. We used a system for doxycycline (dox-inducible target gene expression in metastasis-derived PC3 cells to analyze the effects of hepsin in a quantitative manner. Loss of viability and adhesion correlated with hepsin expression levels during anchorage-dependent but not anchorage-independent growth. Full expression of hepsin led to cell death and detachment and was specifically associated with reduced phosphorylation of AKT at Ser473, which was restored by growth on matrix derived from RWPE1 normal prostatic epithelial cells. In the chorioallantoic membrane xenograft model, hepsin overexpression in PC3 cells reduced the viability of tumors but did not suppress invasive growth. The data presented here provide evidence that elevated levels of hepsin interfere with cell adhesion and viability in the background of prostate cancer as well as other tissue types, the details of which depend on the microenvironment provided. Our findings suggest that overexpression of the enzyme in prostate carcinogenesis must be spatially and temporally restricted for the efficient development of tumors and metastases.

Overexpression of CRF in the BNST diminishes dysphoria but not anxiety-like behavior in nicotine withdrawing rats.

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Qi, Xiaoli; Guzhva, Lidia; Yang, Zhihui; Febo, Marcelo; Shan, Zhiying; Wang, Kevin K W; Bruijnzeel, Adriaan W

2016-09-01

Smoking cessation leads to dysphoria and anxiety, which both increase the risk for relapse. This negative affective state is partly mediated by an increase in activity in brain stress systems. Recent studies indicate that prolonged viral vector-mediated overexpression of stress peptides diminishes stress sensitivity. Here we investigated whether the overexpression of corticotropin-releasing factor (CRF) in the bed nucleus of the stria terminalis (BNST) diminishes nicotine withdrawal symptoms in rats. The effect of nicotine withdrawal on brain reward function was investigated with an intracranial self-stimulation (ICSS) procedure. Anxiety-like behavior was investigated in the elevated plus maze test and a large open field. An adeno-associated virus (AAV) pseudotype 2/5 vector was used to overexpress CRF in the lateral BNST and nicotine dependence was induced using minipumps. Administration of the nicotinic receptor antagonist mecamylamine and cessation of nicotine administration led to a dysphoria-like state, which was prevented by the overexpression of CRF in the BNST. Nicotine withdrawal also increased anxiety-like behavior in the elevated plus maze test and large open field test and slightly decreased locomotor activity in the open field. The overexpression of CRF in the BNST did not prevent the increase in anxiety-like behavior or decrease in locomotor activity. The overexpression of CRF increased CRF1 and CRF2 receptor gene expression and increased the CRF2/CRF1 receptor ratio. In conclusion, the overexpression of CRF in the BNST prevents the dysphoria-like state associated with nicotine withdrawal and increases the CRF2/CRF1 receptor ratio, which may diminish the negative effects of CRF on mood. Published by Elsevier B.V.

Overexpression of rice serotonin N-acetyltransferase 1 in transgenic rice plants confers resistance to cadmium and senescence and increases grain yield.

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Lee, Kyungjin; Back, Kyoungwhan

2017-04-01

While ectopic overexpression of serotonin N-acetyltransferase (SNAT) in plants has been accomplished using animal SNAT genes, ectopic overexpression of plant SNAT genes in plants has not been investigated. Because the plant SNAT protein differs from that of animals in its subcellular localization and enzyme kinetics, its ectopic overexpression in plants would be expected to give outcomes distinct from those observed from overexpression of animal SNAT genes in transgenic plants. Consistent with our expectations, we found that transgenic rice plants overexpressing rice (Oryza sativa) SNAT1 (OsSNAT1) did not show enhanced seedling growth like that observed in ovine SNAT-overexpressing transgenic rice plants, although both types of plants exhibited increased melatonin levels. OsSNAT1-overexpressing rice plants did show significant resistance to cadmium and senescence stresses relative to wild-type controls. In contrast to tomato, melatonin synthesis in rice seedlings was not induced by selenium and OsSNAT1 transgenic rice plants did not show tolerance to selenium. T 2 homozygous OsSNAT1 transgenic rice plants exhibited increased grain yield due to increased panicle number per plant under paddy field conditions. These benefits conferred by ectopic overexpression of OsSNAT1 had not been observed in transgenic rice plants overexpressing ovine SNAT, suggesting that plant SNAT functions differently from animal SNAT in plants. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Specitic gene alterations in radiation-induced tumorigenesis

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Ahn, Joo Mee; Kang, Chang Mo; Lee, Seung Sook; Cho, Chul Koo; Bae, Sang Woo; Lee, Su Jae; Lee, Yun Sil [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2004-07-01

To identify a set of genes involved in the development of radiation-induced tumorigenesis, we used DNA microarrays consisting of 1,176 mouse genes and compared expression profiles of radioresistant cells, designated NIH3T3-R1 and -R4. These cells were tumorigenic in a nude mouse grafting system, as compared to the parental NIH3T3 cells. Expressions of MDM2, CDK6 and CDC25B were found to increase more than 3-fold. Entactin protein levels were downregulated in NIH3T3-R1 and -R4 cells. Changes in expression genes were confirmed by reverse transcription-PCR or western blotting. When these genes were transfected to NIH3T3 cells, the CDC25B and MDM2 overexpressing NIH3T3 cells showed radioresistance, while 2 CDK6 overexpressing cells did not. In the case of entactin overexpressing NIH3T3-R1 or R-4 cells were still radioresistant. Furthermore, the CDC25B and MDM2 overexpressing cells grafted to nude mice, were tumorigenic. NIH3T3-R1 and R4 cells showed increased radiation-induced apoptosis, accompanied by faster growth rate, rather than and earlier radiation-induced G2/M phase arrest, suggesting that the radioresistance of NIH3T3-R1 and R4 cells was due to faster growth rate, rather than induction of apoptosis. In the case of MDM2 and CDC25B overexpressing cells, similar phenomena, such as increased apoptosis and faster growth rate, were shown. The above results, therefore, demonstrate involvement of CDC25B and MDM2 overexpression in radiation-induced tumorigenesis and provide novel targets for detection of radiation-induced carcinogenesis.

Neuroprotective Effect of Ginseng against Alteration of Calcium Binding Proteins Immunoreactivity in the Mice Hippocampus after Radiofrequency Exposure

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Dhiraj Maskey

2013-01-01

Full Text Available Calcium binding proteins (CaBPs such as calbindin D28-k, parvalbumin, and calretinin are able to bind Ca2+ with high affinity. Changes in Ca2+ concentrations via CaBPs can disturb Ca2+ homeostasis. Brain damage can be induced by the prolonged electromagnetic field (EMF exposure with loss of interacellular Ca2+ balance. The present study investigated the radioprotective effect of ginseng in regard to CaBPs immunoreactivity (IR in the hippocampus through immunohistochemistry after one-month exposure at 1.6 SAR value by comparing sham control with exposed and ginseng-treated exposed groups separately. Loss of dendritic arborization was noted with the CaBPs in the Cornu Ammonis areas as well as a decrease of staining intensity of the granule cells in the dentate gyrus after exposure while no loss was observed in the ginseng-treated group. A significant difference in the relative mean density was noted between control and exposed groups but was nonsignificant in the ginseng-treated group. Decrease in CaBP IR with changes in the neuronal staining as observed in the exposed group would affect the hippocampal trisynaptic circuit by alteration of the Ca2+ concentration which could be prevented by ginseng. Hence, ginseng could contribute as a radioprotective agent against EMF exposure, contributing to the maintenance of Ca2+ homeostasis by preventing impairment of intracellular Ca2+ levels in the hippocampus.

Overexpression of CFH gene in pterygiumv patients | Bang | Tropical ...

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Overexpression of CFH gene in pterygiumv patients. Man-Seok Bang, Chang Rae Rho, Bong-Hui Kang, Kyong Jin Cho, Chung-Hun Oh. Abstract. Purpose: To investigate the expression of complement factors in pterygium tissues compared to normal conjunctival samples, using next-generation RNA sequencing. Methods: ...