Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution

DEFF Research Database (Denmark)

Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian

2014-01-01

The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross......-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13); high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13); or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N......-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % (mean (SD)). High-dose Epoetin beta decreased PMI...

The schizophrenia-associated Kv11.1-3.1 isoform results in reduced current accumulation during repetitive brief depolarizations.

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Juliane Heide

Full Text Available Recent genome wide association studies identified a brain and primate specific isoform of a voltage-gated potassium channel, referred to as Kv11.1-3.1, which is significantly associated with schizophrenia. The 3.1 isoform replaces the first 102 amino acids of the most abundant isoform (referred to as Kv11.1-1A with six unique amino acids. Here we show that the Kv11.1-3.1 isoform has faster rates of channel deactivation but a slowing of the rates of inactivation compared to the Kv11.1-1A isoform. The Kv11.1-3.1 isoform also has a significant depolarizing shift in the voltage-dependence of steady-state inactivation. The consequence of the altered gating kinetics is that there is lower current accumulation for Kv11.1-3.1 expressing cells during repetitive action potential firing compared to Kv11.1-1A expressing cells, which in turn will result in longer lasting trains of action potentials. Increased expression of Kv11.1-3.1 channels in the brain of schizophrenia patients might therefore contribute to disorganized neuronal firing.

Biochemical Characteristics of Three Laccase Isoforms from the Basidiomycete Pleurotus nebrodensis

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Xianghe Yuan

2016-02-01

Full Text Available The characterization of three laccase isoforms from Pleurotus nebrodensis is described. Isoenzymes Lac1, Lac2 and Lac3 were purified to homogeneity using ion exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose and a gel filtration step on Superdex 75. The molecular weights of the purified laccases were estimated to be 68, 64 and 51 kDa, respectively. The isoenzymes demonstrated the same optimum pH at 3.0 but slightly different temperature optima: 50–60 °C for Lac1 and Lac3 and 60 °C for Lac2. Lac2 was always more stable than the other two isoforms and exposure to 50 °C for 120 min caused 30% loss in activity. Lac2 was relatively less stable than the other two isoforms when exposed to the pH range of 3.0–8.0 for 24 h, but inactivation only occurred initially, with around 70% residual activity being maintained during the whole process. Oxidative ability towards aromatic compounds varied substantially among the isoforms and each of them displayed preference toward some substrates. Kinetic constants (Km, Kcat were determined by using a 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS assay, with Lac3 showing the best affinity and Lac2 displaying the highest catalytic efficiency. Amino acid sequences from peptides derived from digestion of isoenzymes showed great consistency with laccases in the databases.

Crystallization and Identification of the Glycosylated Moieties of Two Isoforms of the Main Allergen Hev b 2 and Preliminary X-ray Analysis of Two Polymorphs of Isoform ll

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Fuentes-Silva,D.; Mendoza-Hernandez, G.; Stojanoff, V.; Palomares, L.; Zenteno, E.; Torres-Larios, A.; Rodriguez-Romero, A.

2007-01-01

Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a {beta}-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content consisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapor-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4{sub 1} with unit-cell parameters a = b = 150.17, c = 77.41 {angstrom} were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 {angstrom}, {beta}= 113.6{sup o}. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.

Crystallization and identification of the glycosylated moieties of two isoforms of the main allergen Hev b 2 and preliminary X-ray analysis of two polymorphs of isoform II

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Fuentes-Silva, D. [Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Mendoza-Hernández, G. [Facultad de Medicina, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Stojanoff, V. [Brookhaven National Laboratory, National Synchrotron Light Source, Upton, NY (United States); Palomares, L. A. [Instituto de Biotecnología, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Zenteno, E. [Facultad de Medicina, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Torres-Larios, A. [Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Rodríguez-Romero, A., E-mail: adela@servidor.unam.mx [Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico)

2007-09-01

Crystallization of important glycoenzymes involved in IgE-mediated latex allergy. Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a β-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content constisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4{sub 1} with unit-cell parameters a = b = 150.17, c = 77.41 Å were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 Å, β = 113.6°. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.

Crystallization and identification of the glycosylated moieties of two isoforms of the main allergen Hev b 2 and preliminary X-ray analysis of two polymorphs of isoform II

International Nuclear Information System (INIS)

Fuentes-Silva, D.; Mendoza-Hernández, G.; Stojanoff, V.; Palomares, L. A.; Zenteno, E.; Torres-Larios, A.; Rodríguez-Romero, A.

2007-01-01

Crystallization of important glycoenzymes involved in IgE-mediated latex allergy. Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a β-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content constisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4 1 with unit-cell parameters a = b = 150.17, c = 77.41 Å were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 Å, β = 113.6°. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units

Expression of a novel cardiac-specific tropomyosin isoform in humans

International Nuclear Information System (INIS)

Denz, Christopher R.; Narshi, Aruna; Zajdel, Robert W.; Dube, Dipak K.

2004-01-01

Tropomyosins are a family of actin binding proteins encoded by a group of highly conserved genes. Humans have four tropomyosin-encoding genes: TPM1, TPM2, TPM3, and TPM4, each of which is known to generate multiple isoforms by alternative splicing, promoters, and 3 ' end processing. TPM1 is the most versatile and encodes a variety of tissue specific isoforms. The TPM1 isoform specific to striated muscle, designated TPM1α, consists of 10 exons: 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b. In this study, using RT-PCR with adult and fetal human RNAs, we present evidence for the expression of a novel isoform of the TPM1 gene that is specifically expressed in cardiac tissues. The new isoform is designated TPM1κ and contains exon 2a instead of 2b. Ectopic expression of human GFP.TPM1κ fusion protein can promote myofibrillogenesis in cardiac mutant axolotl hearts that are lacking in tropomyosin

Generating Isoform-Specific Antibodies : Lessons from Nucleocytoplasmic Glycoprotein Skp1

NARCIS (Netherlands)

West, Christopher M.; Van Der Wel, Hanke; Chinoy, Zoiesha; Boons, Geert Jan; Gauthier, Ted J.; Taylor, Carol M.; Xu, Yuechi

2015-01-01

Antibodies that discriminate protein isoforms differing by modifications at specific amino acids have revolutionized studies of their functions. Skp1 is a novel nucleocytoplasmic glycoprotein that is hydroxylated at proline-143 and then O-glycosylated by a pentasaccharide attached via a GlcNAcα1,

Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution.

Directory of Open Access Journals (Sweden)

Niels Jacob Aachmann-Andersen

Full Text Available The membrane-assisted isoform immunoassay (MAIIA quantitates erythropoietin (EPO isoforms as percentages of migrated isoforms (PMI. We evaluated the effect of recombinant human EPO (rhEPO on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13; high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13; or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3 % (mean (SD. High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2% (p<0.00001 and 45.2 (7.3% (p<0.00001. Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8% (p<0.00001 and 46.1 (10.4% (p<0.00001. In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4% (p=0.029; low-dose Epoetin beta: 73.1 (17.8% (p=0.039. In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal.

ADAPTIVE CHANGES OF MYOSIN ISOFORMS IN RESPONSE TO LONG-TERM STRENGTH AND POWER TRAINING IN MIDDLE-AGED MEN

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Raivo Puhke

2006-06-01

Full Text Available The purpose of the study was to examine the adaptive changes in myosin heavy chain (MHC and light chain (MLC isoforms in human vastus lateralis muscle caused by long-term strength and power training (54 weeks, approximately 3 times a week in untrained middle- aged men (16 in the training and 6 in the control group. Muscular MHC and MLC isoforms were determined by means of SDS-PAGE gel electrophoresis. During the training period, maximal anaerobic cycling power increased by 64 W (p < 0.001 and the maximal jumping height by 1.5 cm (p < 0. 05 in the training group, but no significant changes were found in the control group. However, the group by time effect was not significant. In the training group, the increase of the maximal jumping height correlated with the number of strength and power training sessions (r = 0.56; p < 0.05. The change of the proportion of MHC IIa isoform from 52.6 ± 12.2% to 59.4 ± 11.6% did not reach statistical significance (p = 0.070 for group by time; within training group p = 0.061 and neither did the change of the proportion of MHC IIx isoform from 18.1 ± 11.4% to 11.1 ± 9.1% (p = 0.104 for group by time; within training group p=0.032. The degree of change of MHC IIx isoform correlated with the amount of earlier recreational sports activity (r = 0.61; p < 0.05. In the training group, the changes of MLC1s isoform correlated negatively with the changes of MLC1f isoform (r = -0. 79; p < 0.05 as well as with the changes in maximal anaerobic cycling power (r = -0.81; p < 0.05, and positively with those of MHC I isoform (r = 0.81; p < 0.05. In conclusion, the long- term strength and power training ~3 times a week seemed to have only slight effects on fast MHC isoforms in the vastus lateralis muscle of untrained middle-aged men; the proportion of MHC IIa tended to increase and that of MHC IIx tended to decrease. No changes in MLC isoform profile could be shown

Comparison of transferrin isoform analysis by capillary electrophoresis and HPLC for screening congenital disorders of glycosylation.

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Dave, Mihika B; Dherai, Alpa J; Udani, Vrajesh P; Hegde, Anaita U; Desai, Neelu A; Ashavaid, Tester F

2018-01-01

Transferrin, a major glycoprotein has different isoforms depending on the number of sialic acid residues present on its oligosaccharide chain. Genetic variants of transferrin as well as the primary (CDG) & secondary glycosylation defects lead to an altered transferrin pattern. Isoform analysis methods are based on charge/mass variations. We aimed to compare the performance of commercially available capillary electrophoresis CDT kit for diagnosing congenital disorders of glycosylation with our in-house optimized HPLC method for transferrin isoform analysis. The isoform pattern of 30 healthy controls & 50 CDG-suspected patients was determined by CE using a Carbohydrate-Deficient Transferrin kit. The results were compared with in-house HPLC-based assay for transferrin isoforms. Transferrin isoform pattern for healthy individuals showed a predominant tetrasialo transferrin fraction followed by pentasialo, trisialo, and disialotransferrin. Two of 50 CDG-suspected patients showed the presence of asialylated isoforms. The results were comparable with isoform pattern obtained by HPLC. The commercial controls showed a <20% CV for each isoform. Bland Altman plot showed the difference plot to be within +1.96 with no systemic bias in the test results by HPLC & CE. The CE method is rapid, reproducible and comparable with HPLC and can be used for screening Glycosylation defects. © 2017 Wiley Periodicals, Inc.

Gene duplication and the evolution of hemoglobin isoform differentiation in birds.

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Grispo, Michael T; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E; Storz, Jay F

2012-11-02

The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the α(A)-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the α(D)-globin gene). The α(D)-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O(2) affinity in the presence of allosteric effectors such as organic phosphates and Cl(-) ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O(2) affinity stems primarily from changes in the O(2) association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the α(D)-globin gene that is shared with the embryonic α-like globin gene.

Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds*

Science.gov (United States)

Grispo, Michael T.; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E.; Storz, Jay F.

2012-01-01

The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the αA-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the αD-globin gene). The αD-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O2 affinity in the presence of allosteric effectors such as organic phosphates and Cl− ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O2 affinity stems primarily from changes in the O2 association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the αD-globin gene that is shared with the embryonic α-like globin gene. PMID:22962007

Casein Kinase I Isoform Hrr25 Is a Negative Regulator of Haa1 in the Weak Acid Stress Response Pathway in Saccharomyces cerevisiae.

Science.gov (United States)

Collins, Morgan E; Black, Joshua J; Liu, Zhengchang

2017-07-01

Haa1 is a transcription factor that adapts Saccharomyces cerevisiae cells to weak organic acid stresses by activating the expression of various genes. Many of these genes encode membrane proteins, such as TPO2 and YRO2 How Haa1 is activated by weak acids is not clear. Here, we show that casein kinase I isoform Hrr25 is an important negative regulator of Haa1. Haa1 is known to be multiply phosphorylated. We found that mutations in HRR25 lead to reduced Haa1 phosphorylation and increased expression of Haa1 target genes and that Hrr25 interacts with Haa1. The other three casein kinase I isoforms, Yck1, Yck2, and Yck3, do not seem to play critical roles in Haa1 regulation. Hrr25 has a 200-residue C-terminal region, including a proline- and glutamine-rich domain. Our data suggest that the C-terminal region of Hrr25 is required for normal inhibition of expression of Haa1 target genes TPO2 and YRO2 and is important for cell growth but is not required for cell morphogenesis. We propose that Hrr25 is an important regulator of cellular adaptation to weak acid stress by inhibiting Haa1 through phosphorylation. IMPORTANCE Our study has revealed the casein kinase I protein Hrr25 to be a negative regulator of Haa1, a transcription factor mediating the cellular response to stresses caused by weak acids. Many studies have focused on the target genes of Haa1 and their roles in weak acid stress responses, but little has been reported on the regulatory mechanism of Haa1. Weak acids, such as acetic acid, have long been used for food preservation by slowing down the growth of fungal species, including S. cerevisiae In the biofuel industry, acetic acid in the lignocellulosic hydrolysates limits the production of ethanol, which is undesirable. By understanding how Haa1 is regulated, we can make advances in the field of food sciences to better preserve food and engineer acetic acid-resistant strains that will increase productivity in the biofuel industry. Copyright © 2017 American

High resolution X-ray structures of mouse major urinary protein nasal isoform in complex with pheromones

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Perez-Miller, Samantha; Zou, Qin; Novotny, Milos V.; Hurley, Thomas D. (Indiana-Med); (Indiana)

2010-09-07

In mice, the major urinary proteins (MUP) play a key role in pheromonal communication by binding and transporting semiochemicals. MUP-IV is the only isoform known to be expressed in the vomeronasal mucosa. In comparison with the MUP isoforms that are abundantly excreted in the urine, MUP-IV is highly specific for the male mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT). To examine the structural basis of this ligand preference, we determined the X-ray crystal structure of MUP-IV bound to three mouse pheromones: SBT, 2,5-dimethylpyrazine, and 2-heptanone. We also obtained the structure of MUP-IV with 2-ethylhexanol bound in the cavity. These four structures show that relative to the major excreted MUP isoforms, three amino acid substitutions within the binding calyx impact ligand coordination. The F103 for A along with F54 for L result in a smaller cavity, potentially creating a more closely packed environment for the ligand. The E118 for G substitution introduces a charged group into a hydrophobic environment. The sidechain of E118 is observed to hydrogen bond to polar groups on all four ligands with nearly the same geometry as seen for the water-mediated hydrogen bond network in the MUP-I and MUP-II crystal structures. These differences in cavity size and interactions between the protein and ligand are likely to contribute to the observed specificity of MUP-IV.

In vivo human apolipoprotein E isoform fractional turnover rates in the CNS.

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Kristin R Wildsmith

Full Text Available Apolipoprotein E (ApoE is the strongest genetic risk factor for Alzheimer's disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4 each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer's disease (AD. Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS, we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis.

[Changes in titin and myosin heavy chain isoform composition in skeletal muscles of Mongolian gerbil (Meriones unguiculatus) after 12-day spaceflight].

Science.gov (United States)

Okuneva, A D; Vikhliantsev, I M; Shpagina, M D; Rogachevskiĭ, V V; Khutsian, S S; Poddubnaia, Z A; Grigor'ev, A I

2012-01-01

Changes of titin and myosin heavy chain isoform composition in skeletal muscles (m. soleus, m. gastrocnemius, m. tibialis anterior, m. psoas major) in Mongolian Gerbil (Meriones unguiculatus ) were investigated after 12-day spaceflight on board of Russian space vehicle "Foton-M3". In m. psoas and m. soleus in the gerbils from "Flight" group the expected increase in the content of fast myosin heavy chain isoforms (IIxd and IIa, respectively) were observed. No significant differences were found in the content of IIxd and IIa isoforms of myosin heavy chain in m. tibialis anterior in the gerbils from control group as compared to that in "Flight" group. An unexpected increase in the content of slow myosin heavy chain I isoform and a decrease in the content of fast IIx/d isoform in m. gastrocnemius of the gerbils from "Flight" group were observed. In skeletal muscles of the gerbils from "Flight" group the relative content of titin N2A-isoform was reduced (by 1,2-1,7 times), although the content of its NT-isoform, which was revealed in striated muscles of mammals in our experiments earlier, remained the same. When the content of titin N2A-isoform was decreased, no predictable abnormalities in sarcomeric structure and contractile ability of skeletal muscles in the gerbils from "Flight" group were found. An assumption on the leading role of titin NT-isoform in maintenance of structural and functional properties of striated muscles of mammals was made.

Cytochrome P450 isoform selectivity in human hepatic theobromine metabolism

Science.gov (United States)

Gates, Simon; Miners, John O

1999-01-01

Aims The plasma clearance of theobromine (TB; 3,7-dimethylxanthine) is known to be induced in cigarette smokers. To determine whether TB may serve as a model substrate for cytochrome P450 (CYP) 1A2, or possibly other isoforms, studies were undertaken to identify the individual human liver microsomal CYP isoforms responsible for the conversion of TB to its primary metabolites. Methods The kinetics of formation of the primary TB metabolites 3-methylxanthine (3-MX), 7-methylxanthine (7-MX) and 3,7-dimethyluric acid (3,7-DMU) by human liver microsomes were characterized using a specific hplc procedure. Effects of CYP isoform-selective xenobiotic inhibitor/substrate probes on each pathway were determined and confirmatory studies with recombinant enzymes were performed to define the contribution of individual isoforms to 3-MX, 7-MX and 3,7-DMU formation. Results The CYP1A2 inhibitor furafylline variably inhibited (0–65%) 7-MX formation, but had no effect on other pathways. Diethyldithiocarbamate and 4-nitrophenol, probes for CYP2E1, inhibited the formation of 3-MX, 7-MX and 3,7-DMU by ≈55–60%, 35–55% and 85%, respectively. Consistent with the microsomal studies, recombinant CYP1A2 and CYP2E1 exhibited similar apparent Km values for 7-MX formation and CYP2E1 was further shown to have the capacity to convert TB to both 3-MX and 3,7-DMU. Conclusions Given the contribution of multiple isoforms to 3-MX and 7-MX formation and the negligible formation of 3,7-DMU in vivo, TB is of little value as a CYP isoform-selective substrate in humans. PMID:10215755

First Trimester Pregnancy Loss and the Expression of alternatively spliced NKp30 isoforms in Maternal Blood and Placental Tissue

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Avishai eShemesh

2015-06-01

Full Text Available In this study, we aimed to investigate whether first trimester pregnancy loss is associated with differences in expression of NKp30 splice variants (isoforms in maternal peripheral blood or placental tissue. We conducted a prospective case-control study; a total of 33 women undergoing dilation and curettage due to first trimester pregnancy loss were further subdivided into groups with sporadic or recurrent pregnancy loss. The control group was comprised of women undergoing elective termination of pregnancy. The qPCR approach was employed to assess the relative expression of NKp30 isoforms as well as the total expression of NKp30 and NKp46 receptors between the selected groups. Results show that in both PBMC and placental tissue, NKp46 and NKp30 expression was mildly elevated in the pregnancy loss groups compared with the elective group. In particular, NKp46 elevation was significant. Moreover, expression analysis of NKp30 isoforms manifested a different profile between PBMC and the placenta. NKp30-a and NKp30-b isoforms in the placental tissue, but not in PBMC, showed a significant increase in the pregnancy loss groups compared with the elective group. Placental expression of NKp30 activating isoforms -a and -b in the pregnancy loss groups was negatively correlated with PLGF expression. In contrast, placental expression of these isoforms in the elective group was positively correlated with TNFα, IL-10 and VEGF-A expression. The altered expression of NKp30 activating isoforms in placental tissue from patients with pregnancy loss compared to the elective group and the different correlations with cytokine expression point to the involvement of NKp30-mediated function in pregnancy loss.

Functional analysis of the isoforms of an ABI3-like factor of Pisum sativum generated by alternative splicing.

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Gagete, Andrés P; Riera, Marta; Franco, Luis; Rodrigo, M Isabel

2009-01-01

At least seven isoforms (PsABI3-1 to PsABI3-7) of a putative, pea ABI3-like factor, originated by alternative splicing, have been identified after cDNA cloning. A similar variability had previously only been described for monocot genes. The full-length isoform, PsABI3-1, contains the typical N-terminal acidic domains and C-terminal basic subdomains, B1 to B3. Reverse transcriptase-PCR analysis revealed that the gene is expressed just in seeds, starting at middle embryogenesis; no gene products are observed in embryo axes after 18 h post-imbibition although they are more persistent in cotyledons. The activity of the isoforms was studied by yeast one-hybrid assays. When yeast was transformed with the isoforms fused to the DNA binding domain of Gal4p, only the polypeptides PsABI3-2 and PsABI3-7 failed to complement the activity of Gal4p. Acidic domains A1 and A2 exhibit transactivating activity, but the former requires a small C-terminal extension to be active. Yeast two-hybrid analysis showed that PsABI3 is able to heterodimerize with Arabidopsis thaliana ABI5, thus proving that PsABI3 is functionally active. The minimum requirement for the interaction PsABI3-AtABI5 is the presence of the subdomain B1 with an extension, 81 amino acids long, at their C-terminal side. Finally, a transient onion transformation assay showed that both the active PsABI3-1 and the inactive PsABI3-2 isoforms are localized to nuclei. Considering that the major isoforms remain approximately constant in developing seeds although their relative proportion varied, the possible role of splicing in the regulatory network of ABA signalling is discussed.

Protein chemical characterization of Gc globulin (vitamin D-binding protein) isoforms; Gc-1f, Gc-1s and Gc-2

DEFF Research Database (Denmark)

Christiansen, Maja; Jørgensen, Charlotte S; Laursen, Inga

2007-01-01

-survival of patients with fulminant hepatic failure and trauma. Here, we characterize the dominant isoforms of plasma-derived Gc globulin from Cohn fraction IV paste with respect to amino acid sequence and posttranslational modifications. Gc globulin was purified in large scale and the isoforms separated by ion...

Structure of ‘linkerless’ hydroxamic acid inhibitor-HDAC8 complex confirms the formation of an isoform-specific subpocket

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Tabackman, Alexa A.; Frankson, Rochelle; Marsan, Eric S.; Perry, Kay; Cole, Kathryn E. (Ithaca); (Cornell); (Christopher Newport U)

2016-11-04

Histone deacetylases (HDACs) catalyze the hydrolysis of acetylated lysine side chains in histone and non-histone proteins, and play a critical role in the regulation of many biological processes, including cell differentiation, proliferation, senescence, and apoptosis. Aberrant HDAC activity is associated with cancer, making these enzymes important targets for drug design. In general, HDAC inhibitors (HDACi) block the proliferation of tumor cells by inducing cell differentiation, cell cycle arrest, and/or apoptosis, and comprise some of the leading therapies in cancer treatments. To date, four HDACi have been FDA approved for the treatment of cancers: suberoylanilide hydroxamic acid (SAHA, Vorinostat, Zolinza®), romidepsin (FK228, Istodax®), belinostat (Beleodaq®), and panobinostat (Farydak®). Most current inhibitors are pan-HDACi, and non-selectively target a number of HDAC isoforms. Six previously reported HDACi were rationally designed, however, to target a unique sub-pocket found only in HDAC8. While these inhibitors were indeed potent against HDAC8, and even demonstrated specificity for HDAC8 over HDACs 1 and 6, there were no structural data to confirm the mode of binding. Here we report the X-ray crystal structure of Compound 6 complexed with HDAC8 to 1.98 Å resolution. We also describe the use of molecular docking studies to explore the binding interactions of the other 5 related HDACi. Our studies confirm that the HDACi induce the formation of and bind in the HDAC8-specific subpocket, offering insights into isoform-specific inhibition.

Isoforms of the major peanut allergen Ara h 2: IgE binding in children with peanut allergy.

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Hales, Belinda J; Bosco, Anthony; Mills, Kristina L; Hazell, Lee A; Loh, Richard; Holt, Patrick G; Thomas, Wayne R

2004-10-01

The major peanut allergen Ara h 2 consists of two isoforms, namely Ara h 2.0101 and Ara h 2.0201. The recently identified Ara h 2.0201 isoform contains an extra 12 amino acids including an extra copy of the reported immunodominant epitope DPYSPS. This study aimed to evaluate the IgE binding of the two Ara h 2 isoforms. Ten clones of Ara h 2 were sequenced to assess the relative frequency of the Ara h 2 isoforms and to identify whether there was further variation in the Ara h 2 sequence. IgE binding to Ara h 2.0101 and Ara h 2.0201 was measured for 70 peanut-allergic children using an IgE DELFIA assay to quantitate specific IgE binding. A competition assay was used to measure whether Ara h 2.0201 contained IgE epitopes other than those found for Ara h 2.0101. The original Ara h 2.0101 sequence was found for 6/10 clones and Ara h 2.0201 was found for 2/10 clones. Ara h 2.0201 had the expected insertion of 12 amino acids as well as substitutions at positions 40 (40G) and 142 (142E). Two new isoforms were identified as different polymorphisms of position 142. One Ara h 2.01 clone (Ara h 2.0102) contained 142E and one Ara h 2.02 clone (Ara h 2.0202) contained 142D. A polymorphism that was previously identified by other investigators at position 77 (77Q or 77R) was not found for any of the 10 sequences. Although the level of IgE binding to Ara h 2.0201 of individual patients was frequently higher than the binding to Ara h 2.0101 (p originally sequenced Ara h 2.0101 isoform and contains other IgE specificities.

Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH isoforms.

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Elaine C Thomas

Full Text Available We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH, a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii communication occurs between the Bateman and catalytic domains and (iii the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

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Guntur, Ananya R.; Gu, Pengyu; Takle, Kendra; Chen, Jingyi; Xiang, Yang; Yang, Chung-Hui

2015-01-01

The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. Here, we show that two specific isoforms of TRPA1 in Drosophila are H2O2 sensitive and that they can detect strong UV light via sensing light-induced production of H2O2. We found that ectopic expression of these H2O2-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H2O2-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H2O2-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. We found that the corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H2O2-sensitive dTRPA1 isoforms and that they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H2O2 production but not conventional phototransduction molecules. Our results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H2O2. We speculate that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response—a function of CC cells—when they encounter strong UV, an aversive stimulus for young larvae. PMID:26443856

Lower glutamic acid decarboxylase 65-kDa isoform messenger RNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia.

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Glausier, Jill R; Kimoto, Sohei; Fish, Kenneth N; Lewis, David A

2015-01-15

Altered gamma-aminobutyric acid (GABA) signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in patients with schizophrenia and schizoaffective disorder. Levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67-kDa isoform (GAD67) in the PFC have been consistently reported to be lower in patients with these disorders, but the status of the second GABA-synthesizing enzyme, glutamic acid decarboxylase 65-kDa isoform (GAD65), remains unclear. GAD65 messenger RNA (mRNA) levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. In a subset of subject pairs, GAD65 relative protein levels were quantified by confocal immunofluorescence microscopy. Mean GAD65 mRNA levels were 13.6% lower in subjects with schizoaffective disorder but did not differ in subjects with schizophrenia relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein levels within subjects with schizoaffective disorder were not attributable to factors commonly comorbid with the diagnosis. In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in patients with schizoaffective disorder relative to patients with schizophrenia, these findings may support an interpretation that GAD65 downregulation provides a homeostatic response complementary to GAD67 downregulation that serves to reduce inhibition in the face of lower PFC network activity. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc

BORIS/CTCFL mRNA isoform expression and epigenetic regulation in epithelial ovarian cancer

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Link, Petra A.; Zhang, Wa; Odunsi, Kunle; Karpf, Adam R.

2013-01-01

Cancer germline (CG) genes are normally expressed in germ cells and aberrantly expressed in a variety of cancers; their immunogenicity has led to the widespread development of cancer vaccines targeting these antigens. BORIS/CTCFL is an autosomal CG antigen and promising cancer vaccine target. BORIS is the only known paralog of CTCF, a gene intimately involved in genomic imprinting, chromatin insulation, and nuclear regulation. We have previously shown that BORIS is expressed in epithelial ovarian cancer (EOC) and that its expression coincides with promoter and global DNA hypomethylation. Recently, 23 different BORIS mRNA variants have been described, and have been functionally grouped into six BORIS isoform families (sf1–sf6). In the present study, we have characterized the expression of BORIS isoform families in normal ovary (NO) and EOC, the latter of which were selected to include two groups with widely varying global DNA methylation status. We find selective expression of BORIS isoform families in NO, which becomes altered in EOC, primarily by the activation of BORIS sf1 in EOC. When comparing EOC samples based on methylation status, we find that BORIS sf1 and sf2 isoform families are selectively activated in globally hypomethylated tumors. In contrast, CTCF is downregulated in EOC, and the ratio of BORIS sf1, sf2, and sf6 isoform families as a function of CTCF is elevated in hypomethylated tumors. Finally, the expression of all BORIS isoform families was induced to varying extents by epigenetic modulatory drugs in EOC cell lines, particularly when DNMT and HDAC inhibitors were used in combination. PMID:23390377

Genomic organization and the tissue distribution of alternatively spliced isoforms of the mouse Spatial gene

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Mattei Marie-Geneviève

2004-07-01

Full Text Available Abstract Background The stromal component of the thymic microenvironment is critical for T lymphocyte generation. Thymocyte differentiation involves a cascade of coordinated stromal genes controlling thymocyte survival, lineage commitment and selection. The "Stromal Protein Associated with Thymii And Lymph-node" (Spatial gene encodes a putative transcription factor which may be involved in T-cell development. In the testis, the Spatial gene is also expressed by round spermatids during spermatogenesis. Results The Spatial gene maps to the B3-B4 region of murine chromosome 10 corresponding to the human syntenic region 10q22.1. The mouse Spatial genomic DNA is organised into 10 exons and is alternatively spliced to generate two short isoforms (Spatial-α and -γ and two other long isoforms (Spatial-δ and -ε comprising 5 additional exons on the 3' site. Here, we report the cloning of a new short isoform, Spatial-β, which differs from other isoforms by an additional alternative exon of 69 bases. This new exon encodes an interesting proline-rich signature that could confer to the 34 kDa Spatial-β protein a particular function. By quantitative TaqMan RT-PCR, we have shown that the short isoforms are highly expressed in the thymus while the long isoforms are highly expressed in the testis. We further examined the inter-species conservation of Spatial between several mammals and identified that the protein which is rich in proline and positive amino acids, is highly conserved. Conclusions The Spatial gene generates at least five alternative spliced variants: three short isoforms (Spatial-α, -β and -γ highly expressed in the thymus and two long isoforms (Spatial-δ and -ε highly expressed in the testis. These alternative spliced variants could have a tissue specific function.

Human HDAC isoform selectivity achieved via exploitation of the acetate release channel with structurally unique small molecule inhibitors

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Whitehead, Lewis; Dobler, Markus R.; Radetich, Branko; Zhu, Yanyi; Atadja, Peter W.; Claiborne, Tavina; Grob, Jonathan E.; McRiner, Andrew; Pancost, Margaret R.; Patnaik, Anup; Shao, Wenlin; Shultz, Michael; Tichkule, Ritesh; Tommasi, Ruben A.; Vash, Brian; Wang, Ping; Stams, Travis (Novartis)

2013-11-20

Herein we report the discovery of a family of novel yet simple, amino-acid derived class I HDAC inhibitors that demonstrate isoform selectivity via access to the internal acetate release channel. Isoform selectivity criteria is discussed on the basis of X-ray crystallography and molecular modeling of these novel inhibitors bound to HDAC8, potentially revealing insights into the mechanism of enzymatic function through novel structural features revealed at the atomic level.

Plasmodium falciparum chloroquine resistance transporter (PfCRT) isoforms PH1 and PH2 perturb vacuolar physiology.

Science.gov (United States)

Callaghan, Paul S; Siriwardana, Amila; Hassett, Matthew R; Roepe, Paul D

2016-03-31

Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT). The approach relies on inducible heterologous expression of PfCRT in Saccharomyces cerevisiae yeast. In these experiments selecting drug concentrations are not toxic to the yeast, nor is expression of PfCRT alone toxic. Only when PfCRT is expressed in the presence of CQ is the growth of yeast impaired, due to inward transport of chloroquine (CQ) via the transporter. During analysis of all 53 known naturally occurring PfCRT isoforms, two isoforms (PH1 and PH2 PfCRT) were found to be intrinsically toxic to yeast, even in the absence of CQ. Additional analysis of six very recently identified PfCRT isoforms from Malaysia also showed some toxicity. In this paper the nature of this yeast toxicity is examined. Data also show that PH1 and PH2 isoforms of PfCRT transport CQ with an efficiency intermediate to that catalyzed by previously studied CQR conferring isoforms. Mutation of PfCRT at position 160 is found to perturb vacuolar physiology, suggesting a fitness cost to position 160 amino acid substitutions. These data further define the wide range of activities that exist for PfCRT isoforms found in P. falciparum isolates from around the globe.

PPARγ isoforms differentially regulate metabolic networks to mediate mouse prostatic epithelial differentiation.

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Strand, D W; Jiang, M; Murphy, T A; Yi, Y; Konvinse, K C; Franco, O E; Wang, Y; Young, J D; Hayward, S W

2012-08-09

Recent observations indicate prostatic diseases are comorbidities of systemic metabolic dysfunction. These discoveries revealed fundamental questions regarding the nature of prostate metabolism. We previously showed that prostate-specific ablation of PPARγ in mice resulted in tumorigenesis and active autophagy. Here, we demonstrate control of overlapping and distinct aspects of prostate epithelial metabolism by ectopic expression of individual PPARγ isoforms in PPARγ knockout prostate epithelial cells. Expression and activation of either PPARγ 1 or 2 reduced de novo lipogenesis and oxidative stress and mediated a switch from glucose to fatty acid oxidation through regulation of genes including Pdk4, Fabp4, Lpl, Acot1 and Cd36. Differential effects of PPARγ isoforms included decreased basal cell differentiation, Scd1 expression and triglyceride fatty acid desaturation and increased tumorigenicity by PPARγ1. In contrast, PPARγ2 expression significantly increased basal cell differentiation, Scd1 expression and AR expression and responsiveness. Finally, in confirmation of in vitro data, a PPARγ agonist versus high-fat diet (HFD) regimen in vivo confirmed that PPARγ agonization increased prostatic differentiation markers, whereas HFD downregulated PPARγ-regulated genes and decreased prostate differentiation. These data provide a rationale for pursuing a fundamental metabolic understanding of changes to glucose and fatty acid metabolism in benign and malignant prostatic diseases associated with systemic metabolic stress.

HPLC separation of human serum albumin isoforms based on their isoelectric points

Science.gov (United States)

Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A.; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

2014-01-01

Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA–SHg+), HSA with Cys34 oxidized to sulfenic acid (HSA–SOH) and HSA oxidized to sulfinate anion (HSA–SO2−) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3–585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA PMID:24316526

Identification of a novel ZIC3 isoform and mutation screening in patients with heterotaxy and congenital heart disease.

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James E J Bedard

Full Text Available Patients with heterotaxy have characteristic cardiovascular malformations, abnormal arrangement of their visceral organs, and midline patterning defects that result from abnormal left-right patterning during embryogenesis. Loss of function of the transcription factor ZIC3 causes X-linked heterotaxy and isolated congenital heart malformations and represents one of the few known monogenic causes of congenital heart disease. The birth incidence of heterotaxy-spectrum malformations is significantly higher in males, but our previous work indicated that mutations within ZIC3 did not account for the male over-representation. Therefore, cross species comparative sequence alignment was used to identify a putative novel fourth exon, and the existence of a novel alternatively spliced transcript was confirmed by amplification from murine embryonic RNA and subsequent sequencing. This transcript, termed Zic3-B, encompasses exons 1, 2, and 4 whereas Zic3-A encompasses exons 1, 2, and 3. The resulting protein isoforms are 466 and 456 amino acid residues respectively, sharing the first 407 residues. Importantly, the last two amino acids in the fifth zinc finger DNA binding domain are altered in the Zic3-B isoform, indicating a potential functional difference that was further evaluated by expression, subcellular localization, and transactivation analyses. The temporo-spatial expression pattern of Zic3-B overlaps with Zic3-A in vivo, and both isoforms are localized to the nucleus in vitro. Both isoforms can transcriptionally activate a Gli binding site reporter, but only ZIC3-A synergistically activates upon co-transfection with Gli3, suggesting that the isoforms are functionally distinct. Screening 109 familial and sporadic male heterotaxy cases did not identify pathogenic mutations in the newly identified fourth exon and larger studies are necessary to establish the importance of the novel isoform in human disease.

Molecular cloning, expression and immunological characterisation of Lol p 5C, a novel allergen isoform of rye grass pollen demonstrating high IgE reactivity.

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Suphioglu, C; Mawdsley, D; Schäppi, G; Gruehn, S; de Leon, M; Rolland, J M; O'Hehir, R E

1999-12-03

A novel isoform of a major rye grass pollen allergen Lol p 5 was isolated from a cDNA expression library. The new isoform, Lol p 5C, shares 95% amino acid sequence identity with Lol p 5A. Both isoforms demonstrated shared antigenic activity but different allergenic activities. Recombinant Lol p 5C demonstrated 100% IgE reactivity in 22 rye grass pollen sensitive patients. In comparison, recombinant Lol p 5A showed IgE reactivity in less than 64% of the patients. Therefore, Lol p 5C represents a novel and highly IgE-reactive isoform allergen of rye grass pollen.

Tumorigenic properties of alternative osteopontin isoforms in mesothelioma

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Ivanov, Sergey V., E-mail: Sergey.Ivanov@med.nyu.edu [Thoracic Surgery Laboratory, Cardiothoracic Surgery Department, NYU Langone Medical Center, 462 First Ave., Bellevue Hospital, Room 15N20, NY 10016 (United States); Ivanova, Alla V.; Goparaju, Chandra M.V.; Chen, Yuanbin; Beck, Amanda; Pass, Harvey I. [Thoracic Surgery Laboratory, Cardiothoracic Surgery Department, NYU Langone Medical Center, 462 First Ave., Bellevue Hospital, Room 15N20, NY 10016 (United States)

2009-05-08

Osteopontin (SPP1) is an inflammatory cytokine that we previously characterized as a diagnostic marker in patients with asbestos-induced malignant mesothelioma (MM). While SPP1 shows both pro- and anti-tumorigenic biological effects, little is known about the molecular basis of these activities. In this study, we demonstrate that while healthy pleura possesses all three differentially spliced SPP1 isoforms (A-C), in clinical MM specimens isoform A is markedly up-regulated and predominant. To provide a clue to possible functions of the SPP1 isoforms we next performed their functional evaluation via transient expression in MM cell lines. As a result, we report that isoforms A-C demonstrate different activities in cell proliferation, wound closure, and invasion assays. These findings suggest different functions for SPP1 isoforms and underline pro-tumorigenic properties of isoforms A and B.

APPRIS 2017: principal isoforms for multiple gene sets

Science.gov (United States)

Rodriguez-Rivas, Juan; Di Domenico, Tomás; Vázquez, Jesús; Valencia, Alfonso

2018-01-01

Abstract The APPRIS database (http://appris-tools.org) uses protein structural and functional features and information from cross-species conservation to annotate splice isoforms in protein-coding genes. APPRIS selects a single protein isoform, the ‘principal’ isoform, as the reference for each gene based on these annotations. A single main splice isoform reflects the biological reality for most protein coding genes and APPRIS principal isoforms are the best predictors of these main proteins isoforms. Here, we present the updates to the database, new developments that include the addition of three new species (chimpanzee, Drosophila melangaster and Caenorhabditis elegans), the expansion of APPRIS to cover the RefSeq gene set and the UniProtKB proteome for six species and refinements in the core methods that make up the annotation pipeline. In addition APPRIS now provides a measure of reliability for individual principal isoforms and updates with each release of the GENCODE/Ensembl and RefSeq reference sets. The individual GENCODE/Ensembl, RefSeq and UniProtKB reference gene sets for six organisms have been merged to produce common sets of splice variants. PMID:29069475

Analysis of human bone alkaline phosphatase isoforms: comparison of isoelectric focusing and ion-exchange high-performance liquid chromatography.

Science.gov (United States)

Sharp, Christopher A; Linder, Cecilia; Magnusson, Per

2007-04-01

Several isoforms of alkaline phosphatase (ALP) can be identified in human tissues and serum after separation by anion-exchange HPLC and isoelectric focusing (IEF). We purified four soluble bone ALP (BALP) isoforms (B/I, B1x, B1 and B2) from human SaOS-2 cells, determined their specific pI values by broad range IEF (pH 3.5-9.5), compared these with commercial preparations of bone, intestinal and liver ALPs and established the effects of neuraminidase and wheat germ lectin (WGA) on enzyme activity. Whilst the isoforms B1x (pI=4.48), B1 (pI=4.32) and B2 (pI=4.12) resolved as well-defined bands, B/I resolved as a complex (pI=4.85-6.84). Neuraminidase altered the migration of all BALP isoforms to pI=6.84 and abolished their binding to the anion-exchange matrix, but increased their enzymatic activities by 11-20%. WGA precipitated the BALP isoforms in IEF gels and the HPLC column and attenuated their enzymatic activities by 54-73%. IEF resolved the commercial BALP into 2 major bands (pI=4.41 and 4.55). Migration of BALP isoforms is similar in IEF and anion-exchange HPLC and dependent on sialic acid content. HPLC is preferable in smaller scale research applications where samples containing mixtures of BALP isoforms are analysed. Circulating liver ALP (pI=3.85) can be resolved from BALP by either method. IEF represents a simpler approach for routine purposes even though some overlapping of the isoforms may occur.

Quarternary structure and enzymological properties of the different hormone-sensitive lipase (HSL) isoforms

DEFF Research Database (Denmark)

Krintel, Christian; Klint, Cecilia; Lindvall, Håkan

2010-01-01

Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of energy in the form of fatty acids from intracellular stores of neutral lipids. The enzyme has been shown to exist in different isoforms with different molecular masses (84 kDa, 89 kDa and 117 kDa) expressed in a tissue-dependen...

Serum concentrations of apelin-17 isoform vary in accordance to blood pressure categories in individuals with obesity class 3.

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Cano Martínez, Luis Javier; Coral Vázquez, Ramón Mauricio; Méndez, Juan Pablo; Trejo, Silvia; Pérez Razo, Juan Carlos; Canto, Patricia

2018-04-13

The aim of this study was to investigate if serum concentrations of apelin-36, apelin-17, apelin-13 or apelin-12 were different in obesity class 3 individuals with hypertension, when compared to those without hypertension (normal or high-normal). Twenty six individuals with obesity class 3-related hypertension and thirty three individuals without hypertension, who were divided in individuals with normal (n = 23) or with high-normal (n = 10) blood pressure (BP) were analyzed. All individuals presented obesity class 3, without diabetes mellitus. Measurements of all apelin isoforms were performed using enzyme-linked immunosorbent assay kits. Analysis of differences between groups of Apelin isoform concentrations was performed by a One-way ANOVA, with a Tukey test post hoc. The individuals of the hypertensive group presented a slightly lower serum concentration of all apelin isoforms, but these differences were not statistically significant. These results were more evident when the group of patients without hypertension were divided based in normal and high-normal BP, observing that apelin-17 isoform were higher in individuals with high-normal BP in comparison to subjects with normal BP (P = 0.018); concentrations were also higher when compared to subjects with hypertension (P = 0.004). To our knowledge, this is the first study regarding the differences of apelin-17 isoform concentrations in individuals pertaining to different categories of BP, who presented obesity class 3. The group of patients that presented hypertension showed a lower concentration of all isoforms. This observation could be due to the fact that these patients were taking antihypertensive medication.

Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions

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Sandy Azzi

2015-06-01

Full Text Available Intrarenal interleukin-15 (IL-15 participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15 isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC to peritumoral (ptumTEC, tumoral (RCC, and cancer stem cells (CSC/CD105+. RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15 isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα. This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105+, where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105+ from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favor tumor progression.

One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

International Nuclear Information System (INIS)

Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A.

2013-01-01

The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility

One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

Energy Technology Data Exchange (ETDEWEB)

Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A., E-mail: roberto.perego@unimib.it

2013-08-01

The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility.

Expression and Immunohistochemical Localisation of the G beta gamma activated and Calcineurin-inhibited Adenylyl Cyclase Isoforms in Rat Articular Chondrocytes

International Nuclear Information System (INIS)

Memon, I.; Khan, K.M.; Siddiqui, S.; Perveen, S.; Ishaq, M.

2016-01-01

Objective: To determine the expression and localisation of the Gβγ-activated adenylyl cyclase (AC) isoforms 2, 4, and 7 and calcineurin-inhibited AC isoform 9 in rat articular chondrocytes. Study Design: Experimental study. Place and Duration of Study: Jumma Research Laboratory and Histology Laboratory, The Aga Khan University, Karachi, from 2009 to 2011. Methodology: Fresh slices of articular cartilage were taken from various synovial joints of rats of different age groups. The expression of AC isoforms was determined by RT-PCR and immunohistochemistry was performed to localise these isoforms in articular chondrocytes. Tissue sections were processed for immunostaining with respective antibodies. The color was developed by diaminobenzidine. Results: All the studied AC isoforms were found to be differentially expressed in different zones of the rat articular cartilage. Generally, expression of all AC isoforms studied increased with age. The expression of the AC isoforms through PCR was almost consistent with the localisation of these isoforms by immunohistochemistry. Conclusion: These data add to the information about signalling cascades possibly involved in articular chondrocytes. Variable expression of AC isoforms 2, 4, 7, and 9 suggest a role for the signalling cascades regulated by the AC isoforms in articular chondrocytes. (author)

The polysaccharide inulin is characterized by an extensive series of periodic isoforms with varying biological actions

Science.gov (United States)

Cooper, Peter D; Barclay, Thomas G; Ginic-Markovic, Milena; Petrovsky, Nikolai

2013-01-01

In studying the molecular basis for the potent immune activity of previously described gamma and delta inulin particles and to assist in production of inulin adjuvants under Good Manufacturing Practice, we identified five new inulin isoforms, bringing the total to seven plus the amorphous form. These isoforms comprise the step-wise inulin developmental series amorphous → alpha-1 (AI-1) → alpha-2 (AI-2) → gamma (GI) → delta (DI) → zeta (ZI) → epsilon (EI) → omega (OI) in which each higher isoform can be made either by precipitating dissolved inulin or by direct conversion from its precursor, both cases using regularly increasing temperatures. At higher temperatures, the shorter inulin polymer chains are released from the particle and so the key difference between isoforms is that each higher isoform comprises longer polymer chains than its precursor. An increasing trend of degree of polymerization is confirmed by end-group analysis using 1H nuclear magnetic resonance spectroscopy. Inulin isoforms were characterized by the critical temperatures of abrupt phase-shifts (solubilizations or precipitations) in water suspensions. Such (aqueous) “melting” or “freezing” points are diagnostic and occur in strikingly periodic steps reflecting quantal increases in noncovalent bonding strength and increments in average polymer lengths. The (dry) melting points as measured by modulated differential scanning calorimetry similarly increase in regular steps. We conclude that the isoforms differ in repeated increments of a precisely repeating structural element. Each isoform has a different spectrum of biological activities and we show the higher inulin isoforms to be more potent alternative complement pathway activators. PMID:23853206

The C/EBPbeta isoform, liver-inhibitory protein (LIP), induces autophagy in breast cancer cell lines

International Nuclear Information System (INIS)

Abreu, Maria M.; Sealy, Linda

2010-01-01

Autophagy is a process involving the bulk degradation of cellular components in the cytoplasm via the lysosomal degradation pathway. Autophagy manifests a protective role in stressful conditions such as nutrient or growth factor depletion; however, extensive degradation of regulatory molecules or organelles essential for survival can lead to the demise of the cell, or autophagy-mediated cell death. The role of autophagy in cancer is complex with roles in both tumor suppression and tumor promotion proposed. Here we report that an isoform of the C/EBPbeta transcription factor, liver-enriched inhibitory protein (LIP), induces cell death in human breast cancer cells and stimulates autophagy. Overexpression of LIP is incompatible with cell growth and when cell cycle analysis was performed, a DNA profile of cells undergoing apoptosis was not observed. Instead, LIP expressing cells appeared to have large autophagic vesicles when examined via electron microscopy. Autophagy was further assessed in LIP expressing cells by monitoring the development of acidic vesicular organelles and conversion of LC3 from the cytoplasmic form to the membrane-bound form. Our work shows that C/EBPbeta isoform, LIP, is another member of the group of transcription factors, including E2F1 and p53, which are capable of playing a role in autophagy.

The C/EBPbeta isoform, liver-inhibitory protein (LIP), induces autophagy in breast cancer cell lines

Energy Technology Data Exchange (ETDEWEB)

Abreu, Maria M. [Department of Cancer Biology, 752 Preston Research Building, Vanderbilt University, Nashville, TN 37232 (United States); Sealy, Linda, E-mail: Linda.sealy@vanderbilt.edu [Department of Cancer Biology, 752 Preston Research Building, Vanderbilt University, Nashville, TN 37232 (United States); Department of Molecular Physiology and Biophysics, 752 Preston Research Building, Vanderbilt University, Nashville, TN 37232 (United States)

2010-11-15

Autophagy is a process involving the bulk degradation of cellular components in the cytoplasm via the lysosomal degradation pathway. Autophagy manifests a protective role in stressful conditions such as nutrient or growth factor depletion; however, extensive degradation of regulatory molecules or organelles essential for survival can lead to the demise of the cell, or autophagy-mediated cell death. The role of autophagy in cancer is complex with roles in both tumor suppression and tumor promotion proposed. Here we report that an isoform of the C/EBPbeta transcription factor, liver-enriched inhibitory protein (LIP), induces cell death in human breast cancer cells and stimulates autophagy. Overexpression of LIP is incompatible with cell growth and when cell cycle analysis was performed, a DNA profile of cells undergoing apoptosis was not observed. Instead, LIP expressing cells appeared to have large autophagic vesicles when examined via electron microscopy. Autophagy was further assessed in LIP expressing cells by monitoring the development of acidic vesicular organelles and conversion of LC3 from the cytoplasmic form to the membrane-bound form. Our work shows that C/EBPbeta isoform, LIP, is another member of the group of transcription factors, including E2F1 and p53, which are capable of playing a role in autophagy.

Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein?

Directory of Open Access Journals (Sweden)

Piero Leone

2018-01-01

Full Text Available FAD synthase (FADS, EC 2.7.7.2 is the last essential enzyme involved in the pathway of biosynthesis of Flavin cofactors starting from Riboflavin (Rf. Alternative splicing of the human FLAD1 gene generates different isoforms of the enzyme FAD synthase. Besides the well characterized isoform 1 and 2, other FADS isoforms with different catalytic domains have been detected, which are splice variants. We report the characterization of one of these novel isoforms, a 320 amino acid protein, consisting of the sole C-terminal 3′-phosphoadenosine 5′-phosphosulfate (PAPS reductase domain (named FADS6. This isoform has been previously detected in Riboflavin-Responsive (RR-MADD and Non-responsive Multiple Acyl-CoA Dehydrogenase Deficiency (MADD patients with frameshift mutations of FLAD1 gene. To functionally characterize the hFADS6, it has been over-expressed in Escherichia coli and purified with a yield of 25 mg·L−1 of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (kcat about 2.8 min−1, as well as FAD pyrophosphorolysis in a strictly Mg2+-dependent manner. The synthesis of FAD is inhibited by HgCl2. The enzyme lacks the ability to hydrolyze FAD. It behaves similarly to PAPS. Combining threading and ab-initio strategy a 3D structural model for such isoform has been built. The relevance to human physio-pathology of this FADS isoform is discussed.

Photoperiodism and crassulacean acid metabolism : I. Immunological and kinetic evidences for different patterns of phosphoenolpyruvate carboxylase isoforms in photoperiodically inducible and non-inducible Crassulacean acid metabolism plants.

Science.gov (United States)

Brulfert, J; Müller, D; Kluge, M; Queiroz, O

1982-05-01

Plants of Kalanchoe blossfeldiana v. Poelln. Tom Thumb and Sedum morganianum E. Walth. were grown under controlled photoperiodic conditions under either short or long days. Gaz exchange measurements confirmed that in K. blossfeldiana Crassulacean acid metabolism (CAM) was photoperiodically inducible and that S. morganianum performed CAM independently of photoperiod. With K. blossfeldiana, a comparison of catalytic and regulatory properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) from short-day and long-day grown plants showed differences, but not with S. morganianum. Ouchterlony double diffusion tests and immunotitration experiments (using a S. morganianum PEPC antibody) established that CAM is induced in K. blossfeldiana-but not in S. morganianum-through the synthesis of a new PEPC isoform; this form shows an immunological behavior different from that prevailing under non-inductive conditions and can be considered as specific for CAM performance.

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

International Nuclear Information System (INIS)

Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria

2006-01-01

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl 2 , as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 ± 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K M value for FMN of 1.5 ± 0.3 μM. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast

Expression of the human isoform of glutamate dehydrogenase, hGDH2, augments TCA cycle capacity and oxidative metabolism of glutamate during glucose deprivation in astrocytes

DEFF Research Database (Denmark)

Nissen, Jakob D; Lykke, Kasper; Bryk, Jaroslaw

2017-01-01

A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1...... and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation...

WT1 isoform expression pattern in acute myeloid leukemia.

Science.gov (United States)

Luna, Irene; Such, Esperanza; Cervera, Jose; Barragán, Eva; Ibañez, Mariam; Gómez-Seguí, Inés; López-Pavía, María; Llop, Marta; Fuster, Oscar; Dolz, Sandra; Oltra, Silvestre; Alonso, Carmen; Vera, Belén; Lorenzo, Ignacio; Martínez-Cuadrón, David; Montesinos, Pau; Senent, M Leonor; Moscardó, Federico; Bolufer, Pascual; Sanz, Miguel A

2013-12-01

WT1 plays a dual role in leukemia development, probably due to an imbalance in the expression of the 4 main WT1 isoforms. We quantify their expression and evaluate them in a series of AML patients. Our data showed a predominant expression of isoform D in AML, although in a lower quantity than in normal CD34+ cells. We found a positive correlation between the total WT1 expression and A, B and C isoforms. The overexpression of WT1 in AML might be due to a relative increase in A, B and C isoforms, together with a relative decrease in isoform D expression. Copyright © 2013 Elsevier Ltd. All rights reserved.

Purification of a new isoform of laccase from a Marasmius quercophilus strain isolated from a cork oak litter (Quercus suber L).

Science.gov (United States)

Farnet, A M; Criquet, S; Pocachard, E; Gil, G; Ferre, E

2002-01-01

A new isoform of laccase from Marasmius quercophilus is described in this study. The strain of this white-rot fungus was isolated for the first time on a cork oak litter. This isoform exhibited certain common properties of laccases (a molecular weight of 65 Kda, an optimum pH of 6.2 with syringaldazine). But this laccase has also particularly novel features: the best activity measured was observed at high temperatures (80 C) and this isoform was not inhibited with EDTA. Furthermore, this induced laccase was able to transform most of the aromatic compounds tested without the addition of mediators to the reaction mixture, and the transformation of certain chlorophenols (2-chlorophenol and 2,4-dichlorophenol) by a laccase isoform from M. quercophilus is reported here for the first time. We also demonstrate the importance of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as a mediator since it allowed veratryl alcohol and p-hydroxybenzoic acid transformation. Moreover, new products of transformation were observed using the combination of ABTS with this isoform of laccase.

Characterisation of Cdkl5 transcript isoforms in rat.

Science.gov (United States)

Hector, Ralph D; Dando, Owen; Ritakari, Tuula E; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

2017-03-01

CDKL5 deficiency is a severe neurological disorder caused by mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5). The predominant human CDKL5 brain isoform is a 9.7kb transcript comprised of 18 exons with a large 6.6kb 3'-untranslated region (UTR). Mammalian models of CDKL5 disorder are currently limited to mouse, and little is known about Cdkl5 in other organisms used to model neurodevelopmental disorders, such as rat. In this study we characterise, both bioinformatically and experimentally, the rat Cdkl5 gene structure and its associated transcript isoforms. New exonic regions, splice sites and UTRs are described, confirming the presence of four distinct transcript isoforms. The predominant isoform in the brain, which we name rCdkl5_1, is orthologous to the human hCDKL5_1 and mouse mCdkl5_1 isoforms and is the most highly expressed isoform across all brain regions tested. This updated gene model of Cdkl5 in rat provides a framework for studies into its protein products and provides a reference for the development of molecular therapies for testing in rat models of CDKL5 disorder. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of the synaptotagmin family during reconstituted membrane fusion. Uncovering a class of inhibitory isoforms.

Science.gov (United States)

Bhalla, Akhil; Chicka, Michael C; Chapman, Edwin R

2008-08-01

Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells is regulated by the Ca(2+)-binding protein synaptotagmin (syt) I. Sixteen additional isoforms of syt have been identified, but little is known concerning their biochemical or functional properties. Here, we assessed the abilities of fourteen syt isoforms to directly regulate SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor)-catalyzed membrane fusion. One group of isoforms stimulated neuronal SNARE-mediated fusion in response to Ca(2+), while another set inhibited SNARE catalyzed fusion in both the absence and presence of Ca(2+). Biochemical analysis revealed a strong correlation between the ability of syt isoforms to bind 1,2-dioleoyl phosphatidylserine (PS) and t-SNAREs in a Ca(2+)-promoted manner with their abilities to enhance fusion, further establishing PS and SNAREs as critical effectors for syt action. The ability of syt I to efficiently stimulate fusion was specific for certain SNARE pairs, suggesting that syts might contribute to the specificity of intracellular membrane fusion reactions. Finally, a subset of inhibitory syts down-regulated the ability of syt I to activate fusion, demonstrating that syt isoforms can modulate the function of each other.

Oxygenation properties and isoform diversity of snake hemoglobins

DEFF Research Database (Denmark)

Storz, Jay F.; Natarajan, Chandrasekhar; Moriyama, Hideaki

2015-01-01

Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer- dimer dissociation. However, standardized comparative data are lacking fo...... isoform of the South American rattlesnake is homologous to the minor HbD of other amniotes and, contrary to the pattern of Hb isoform differentiation in birds and turtles, exhibits a lower O2 affinity than the HbA isoform....

Different expression patterns of renal Na+/K+-ATPase α-isoform-like proteins between tilapia and milkfish following salinity challenges.

Science.gov (United States)

Yang, Wen-Kai; Chung, Chang-Hung; Cheng, Hui Chen; Tang, Cheng-Hao; Lee, Tsung-Han

2016-12-01

Euryhaline teleosts can survive in a broad range of salinity via alteration of the molecular mechanisms in certain osmoregulatory organs, including in the gill and kidney. Among these mechanisms, Na + /K + -ATPase (NKA) plays a crucial role in triggering ion-transporting systems. The switch of NKA isoforms in euryhaline fish gills substantially contributes to salinity adaptation. However, there is little information about switches in the kidneys of euryhaline teleosts. Therefore, the responses of the renal NKA α-isoform protein switch to salinity challenge in euryhaline tilapia (Oreochromis mossambicus) and milkfish (Chanos chanos) with different salinity preferences were examined and compared in this study. Immunohistochemical staining in tilapia kidneys revealed the localization of NKA in renal tubules rather than in the glomeruli, similar to our previous findings in milkfish kidneys. Protein abundance in the renal NKA pan α-subunit-like, α1-, and α3-isoform-like proteins in seawater-acclimated tilapia was significantly higher than in the freshwater group, whereas the α2-isoform-like protein exhibited the opposite pattern of expression. In the milkfish, higher protein abundance in the renal NKA pan α-subunit-like and α1-isoform-like proteins was found in freshwater-acclimated fish, whereas no difference was found in the protein abundance of α2- and α3-isoform-like proteins between groups. These findings suggested that switches for renal NKA α-isoforms, especially the α1-isoform, were involved in renal osmoregulatory mechanisms of euryhaline teleosts. Moreover, differences in regulatory responses of the renal NKA α-subunit to salinity acclimation between tilapia and milkfish revealed that divergent mechanisms for maintaining osmotic balance might be employed by euryhaline teleosts with different salinity preferences. Copyright © 2016 Elsevier Inc. All rights reserved.

Pre-α-pro-GDNF and Pre-β-pro-GDNF Isoforms Are Neuroprotective in the 6-hydroxydopamine Rat Model of Parkinson's Disease

Directory of Open Access Journals (Sweden)

Anna-Maija Penttinen

2018-06-01

Full Text Available Glial cell line-derived neurotrophic factor (GDNF is one of the most studied neurotrophic factors. GDNF has two splice isoforms, full-length pre-α-pro-GDNF (α-GDNF and pre-β-pro-GDNF (β-GDNF, which has a 26 amino acid deletion in the pro-region. Thus far, studies have focused solely on the α-GDNF isoform, and nothing is known about the in vivo effects of the shorter β-GDNF variant. Here we compare for the first time the effects of overexpressed α-GDNF and β-GDNF in non-lesioned rat striatum and the partial 6-hydroxydopamine lesion model of Parkinson's disease. GDNF isoforms were overexpressed with their native pre-pro-sequences in the striatum using an adeno-associated virus (AAV vector, and the effects on motor performance and dopaminergic phenotype of the nigrostriatal pathway were assessed. In the non-lesioned striatum, both isoforms increased the density of dopamine transporter-positive fibers at 3 weeks after viral vector delivery. Although both isoforms increased the activity of the animals in cylinder assay, only α-GDNF enhanced the use of contralateral paw. Four weeks later, the striatal tyrosine hydroxylase (TH-immunoreactivity was decreased in both α-GDNF and β-GDNF treated animals. In the neuroprotection assay, both GDNF splice isoforms increased the number of TH-immunoreactive cells in the substantia nigra but did not promote behavioral recovery based on amphetamine-induced rotation or cylinder assays. Thus, the shorter GDNF isoform, β-GDNF, and the full-length α-isoform have comparable neuroprotective efficacy on dopamine neurons of the nigrostriatal circuitry.

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription

DEFF Research Database (Denmark)

Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien

2012-01-01

site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial...... diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites...... and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain...

The characterization of soybean oil body integral oleosin isoforms and the effects of alkaline pH on them.

Science.gov (United States)

Cao, Yanyun; Zhao, Luping; Ying, Yusang; Kong, Xiangzhen; Hua, Yufei; Chen, Yeming

2015-06-15

Oil body, an organelle in seed cell (naturally pre-emulsified oil), has great potentials to be used in food, cosmetics, pharmaceutical and other applications requiring stable oil-in-water emulsions. Researchers have tried to extract oil body by alkaline buffers, which are beneficial for removing contaminated proteins. But it is not clear whether alkaline buffers could remove oil body integral proteins (mainly oleosins), which could keep oil body integrity and stability. In this study, seven oleosin isoforms were identified for soybean oil body (three isoforms, 24 kDa; three isoforms, 18 kDa; one isoform, 16kDa). Oleosins were not glycoproteins and 24 kDa oleosin isoforms possessed less thiol groups than 18 kDa ones. It was found that alkaline pH not only removed contaminated proteins but also oleosins, and more and more oleosins were removed with increasing alkaline pH. Copyright © 2015 Elsevier Ltd. All rights reserved.

Diagnostic Accuracy of Cerebrospinal Fluid Amyloid-β Isoforms for Early and Differential Dementia Diagnosis.

Science.gov (United States)

Struyfs, Hanne; Van Broeck, Bianca; Timmers, Maarten; Fransen, Erik; Sleegers, Kristel; Van Broeckhoven, Christine; De Deyn, Peter P; Streffer, Johannes R; Mercken, Marc; Engelborghs, Sebastiaan

2015-01-01

Overlapping cerebrospinal fluid biomarkers (CSF) levels between Alzheimer's disease (AD) and non-AD patients decrease differential diagnostic accuracy of the AD core CSF biomarkers. Amyloid-β (Aβ) isoforms might improve the AD versus non-AD differential diagnosis. To determine the added diagnostic value of Aβ isoforms, Aβ(1-37), Aβ(1-38), and Aβ(1-40), as compared to the AD CSF biomarkers Aβ(1-42), T-tau, and P-tau(181P). CSF from patients with dementia due to AD (n = 50), non-AD dementias (n = 50), mild cognitive impairment due to AD (n = 50) and non-demented controls (n = 50) was analyzed with a prototype multiplex assay using MSD detection technology. The non-AD group consisted of frontotemporal dementia (FTD; n = 17), dementia with Lewy bodies (DLB; n = 17), and vascular dementia (n = 16). Aβ(1-37) and Aβ(1-38) increased accuracy to differentiate AD from FTD or DLB. Aβ(1-37), Aβ(1-38), and Aβ(1-40) levels correlated with Mini-Mental State Examination scores and disease duration in dementia due to AD. The Aβ(1-42)/Aβ(1-40) ratio improved diagnostic performance of Aβ(1-42) in most differential diagnostic situations. Aβ(1-42) levels were lower in APOE ε4 carriers compared to non-carriers. Aβ isoforms help to differentiate AD from FTD and DLB. Aβ isoforms increase diagnostic performance of Aβ(1-42). In contrast to Aβ1-42, Aβ isoforms seem to be correlated with disease severity in AD. Adding the Aβ isoforms to the current biomarker panel could enhance diagnostic accuracy.

Identification and characterization of novel smoothelin isoforms in vascular smooth muscle.

Science.gov (United States)

Krämer, J; Quensel, C; Meding, J; Cardoso, M C; Leonhardt, H

2001-01-01

Smoothelin is a cytoskeletal protein specifically expressed in differentiated smooth muscle cells and has been shown to colocalize with smooth muscle alpha actin. In addition to the small smoothelin isoform of 59 kD, we recently identified a large smoothelin isoform of 117 kD. The aim of this study was to identify and characterize novel smoothelin isoforms. The genomic structure and sequence of the smoothelin gene were determined by genomic PCR, RT-PCR and DNA sequencing. Comparison of the cDNA and genomic sequences shows that the small smoothelin isoform is generated by transcription initiation 10 kb downstream of the start site of the large isoform. In addition to the known smoothelin cDNA (c1 isoform) we identified two novel cDNA variants (c2 and c3 isoform) that are generated by alternative splicing within a region, which shows similarity to the spectrin family of F-actin cross-linking proteins. Visceral organs express the c1 form, while the c2 form prevails in well-vascularized tissue as analyzed by RT-PCR. We then generated specific antibodies against the major smoothelin isoforms and could show by Western blotting and immunohistochemistry that the large isoform is specifically expressed in vascular smooth muscle cells, while the small isoform is abundant in visceral smooth muscle. These results strongly suggest that the smoothelin gene contains a vascular and a visceral smooth muscle promoter. The cell-type-specific expression of smoothelin isoforms that are associated with actin filaments may play a role in the modulation of the contractile properties of different smooth muscle cell types. Copyright 2001 S. Karger AG, Basel

Inulin isoforms differ by repeated additions of one crystal unit cell

Science.gov (United States)

Cooper, Peter D.; Barclay, Thomas G.; Ginic-Markovic, Milena; Gerson, Andrea R.; Petrovsky, Nikolai

2014-01-01

Inulin isoforms, especially delta inulin, are important biologically as immune activators and clinically as vaccine adjuvants. In exploring action mechanisms, we previously found regular increments in thermal properties of the seven-member inulin isoform series that suggested regular additions of some energetic structural unit. Because the previous isolates carried additional longer chains that masked defining ranges, these were contrasted with new isoform isolates comprising only inulin chain lengths defining that isoform. The new series began with 19 fructose units per chain (alpha-1 inulin), increasing regularly by 6 fructose units per isoform. Thus the ‘energetic unit’ equates to 6 fructose residues per chain. All isoforms showed indistinguishable X-ray diffraction patterns that were also identical with known inulin crystals. We conclude that an ‘energetic unit’ equates to one helix turn of 6 fructose units per chain as found in one unit cell of the inulin crystal. Each isoform chain comprised progressively more helix turns plus one additional fructose and glucose residues per chain. PMID:24528745

Prostaglandin D Synthase Isoforms from Cerebrospinal Fluid Vary with Brain Pathology

Directory of Open Access Journals (Sweden)

Michael G. Harrington

2006-01-01

Full Text Available Glutathione independent prostaglandin D synthase (Swissprot P41222, PTGDS has been identified in human cerebrospinal fluid and some changes in PTGDS in relation to disease have been reported. However, little is known of the extent that PTGDS isoforms fluctuate across a large range of congenital and acquired diseases. The purpose of this study was to examine changes in PTGDS isoforms in such a population. Spinal fluid from 22 healthy study participants (normal controls with no classifiable neurological or psychiatric diagnosis was obtained and PTGDS isoforms were identified by specific immunostaining and mass spectrometry after denaturing 2D gel electrophoresis. The PTGDS isoforms in controls consisted of five charge isoforms that were always present and a small number of occasional, low abundance isoforms. A qualitative survey of 98 different people with a wide range of congenital and acquired diseases revealed striking changes. Loss of the control isoforms occurred in congenital malformations of the nervous system. Gain of additional isoforms occurred in some degenerative, most demyelinating and vasculitic diseases, as well as in Creutzfeldt-Jakob disease. A retrospective analysis of published data that quantified relative amounts of PTGDS in multiple sclerosis, schizophrenia and Parkinson’s disease compared to controls revealed significant dysregulation. It is concluded that qualitative and quantitative fluctuations of cerebrospinal fluid PTGDS isoforms reflect both major and subtle brain pathophysiology.

Identification of signals that facilitate isoform specific nucleolar localization of myosin IC

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Schwab, Ryan S.; Ihnatovych, Ivanna; Yunus, Sharifah Z.S.A.; Domaradzki, Tera [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States); Hofmann, Wilma A., E-mail: whofmann@buffalo.edu [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States)

2013-05-01

Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosin IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms. - Highlights: ► Two NoLS have been identified in the myosin IC isoform B sequence. ► Both NoLS are necessary for myosin IC isoform B specific nucleolar localization. ► First mechanistic explanation of functional differences between the isoforms.

Modulation of neuronal differentiation by CD40 isoforms

International Nuclear Information System (INIS)

Hou Huayu; Obregon, Demian; Lou, Deyan; Ehrhart, Jared; Fernandez, Frank; Silver, Archie; Tan Jun

2008-01-01

Neuron differentiation is a complex process involving various cell-cell interactions, and multiple signaling pathways. We showed previously that CD40 is expressed and functional on mouse and human neurons. In neurons, ligation of CD40 protects against serum withdrawal-induced injury and plays a role in survival and differentiation. CD40 deficient mice display neuron dysfunction, aberrant neuron morphologic changes, and associated gross brain abnormalities. Previous studies by Tone and colleagues suggested that five isoforms of CD40 exist with two predominant isoforms expressed in humans: signal-transducible CD40 type I and a C-terminal truncated, non-signal-transducible CD40 type II. We hypothesized that differential expression of CD40 isoform type I and type II in neurons may modulate neuron differentiation. Results show that adult wild-type, and CD40 -/- deficient mice predominantly express CD40 type I and II isoforms. Whereas adult wild-type mice express mostly CD40 type I in cerebral tissues at relatively high levels, in age and gender-matched CD40 -/- mice CD40 type I expression was almost completely absent; suggesting a predominance of the non-signal-transducible CD40 type II isoform. Younger, 1 day old wild-type mice displayed less CD40 type I, and more CD40 type II, as well as, greater expression of soluble CD40 (CD40L/CD40 signal inhibitor), compared with 1 month old mice. Neuron-like N2a cells express CD40 type I and type II isoforms while in an undifferentiated state, however once induced to differentiate, CD40 type I predominates. Further, differentiated N2a cells treated with CD40 ligand express high levels of neuron specific nuclear protein (NeuN); an effect reduced by anti-CD40 type I siRNA, but not by control (non-targeting) siRNA. Altogether these data suggest that CD40 isoforms may act in a temporal fashion to modulate neuron differentiation during brain development. Thus, modulation of neuronal CD40 isoforms and CD40 signaling may represent

Comparison of folic acid levels in schizophrenic patients and control groups

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Arthy, C. C.; Amin, M. M.; Effendy, E.

2018-03-01

Folic acid deficiency is a risk factor for schizophrenia through epidemiology, biochemistry and gene-related studies. Compared with healthy people, schizophrenic patients may have high homocysteine plasma values and homocysteine or low levels of folic acid, which seems to correlate with extrapyramidal motor symptoms caused by neuroleptic therapy and with symptoms of schizophrenia. In this present study, we focus on the difference of folic acid level between schizophrenic patient and control group. The study sample consisted of schizophrenic patients and 14 people in the control group and performed blood sampling to obtain the results of folic acid levels. The folic acid level in both groups was within normal range, but the schizophrenic patient group had lower mean folic acid values of 5.00 ng/ml (sb 1.66), compared with the control group with mean folic acid values of 10.75 ng/ml (sb 4.33). there was the group of the control group had a higher value of folic acid than the schizophrenic group.

Detection of VEGF-A(xxx)b isoforms in human tissues.

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Bates, David O; Mavrou, Athina; Qiu, Yan; Carter, James G; Hamdollah-Zadeh, Maryam; Barratt, Shaney; Gammons, Melissa V; Millar, Ann B; Salmon, Andrew H J; Oltean, Sebastian; Harper, Steven J

2013-01-01

Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.

Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle [version 2; referees: 3 approved

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Christine Chaponnier

2016-06-01

Full Text Available Higher vertebrates (mammals and birds express six different highly conserved actin isoforms that can be classified in three subgroups: 1 sarcomeric actins, α-skeletal (α-SKA and α-cardiac (α-CAA, 2 smooth muscle actins (SMAs, α-SMA and γ-SMA, and 3 cytoplasmic actins (CYAs, β-CYA and γ-CYA. The variations among isoactins, in each subgroup, are due to 3-4 amino acid differences located in their acetylated N-decapeptide sequence. The first monoclonal antibody (mAb against an actin isoform (α-SMA was produced and characterized in our laboratory in 1986 (Skalli  et al., 1986 . We have further obtained mAbs against the 5 other isoforms. In this report, we focus on the mAbs anti-α-SKA and anti-α-CAA obtained after immunization of mice with the respective acetylated N-terminal decapeptides using the Repetitive Immunizations at Multiple Sites Strategy (RIMMS. In addition to the identification of their epitope by immunoblotting, we describe the expression of the 2 sarcomeric actins in mature skeletal muscle and during muscle repair after micro-lesions. In particular, we analyze the expression of α-CAA, α-SKA and α-SMA by co-immunostaining in a time course frame during the muscle repair process. Our results indicate that a restricted myocyte population expresses α-CAA and suggest a high capacity of self-regeneration in muscle cells. These antibodies may represent a helpful tool for the follow-up of muscle regeneration and pathological changes.

Selectivity analyses of γ-benzylidene digoxin derivatives to different Na,K-ATPase α isoforms: a molecular docking approach.

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Pessôa, Marco T C; Alves, Silmara L G; Taranto, Alex G; Villar, José A F P; Blanco, Gustavo; Barbosa, Leandro A

2018-12-01

Digoxin and other cardiotonic steroids (CTS) exert their effect by inhibiting Na,K-ATPase (NKA) activity. CTS bind to the various NKA isoforms that are expressed in different cell types, which gives CTS their narrow therapeutic index. We have synthesised a series of digoxin derivatives (γ-Benzylidene digoxin derivatives) with substitutions in the lactone ring (including non-oxygen and ether groups), to obtain CTS with better NKA isoform specificity. Some of these derivatives show some NKA isoform selective effects, with BD-3, BD-8, and BD-13 increasing NKA α2 activity, BD-5 inhibiting NKA α1 and NKA α3, BD-10 reducing NKA α1, but stimulating NKA α2 and α3; and BD-14, BD-15, and BD-16 enhancing NKA α3 activity. A molecular-docking approach favoured NKA isoform specific interactions for the compounds that supported their observed activity. These results show that BD compounds are a new type of CTS with the capacity to target NKA activity in an isoform-specific manner.

Regulation of cardiac remodeling by cardiac Na/K-ATPase isoforms

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Lijun Catherine Liu

2016-09-01

Full Text Available Cardiac remodeling occurs after cardiac pressure/volume overload or myocardial injury during the development of heart failure and is a determinant of heart failure. Preventing or reversing remodeling is a goal of heart failure therapy. Human cardiomyocyte Na+/K+-ATPase has multiple α isoforms (1-3. The expression of the α subunit of the Na+/K+-ATPase is often altered in hypertrophic and failing hearts. The mechanisms are unclear. There are limited data from human cardiomyocytes. Abundant evidences from rodents show that Na+/K+-ATPase regulates cardiac contractility, cell signaling, hypertrophy and fibrosis. The α1 isoform of the Na+/K+-ATPase is the ubiquitous isoform and possesses both pumping and signaling functions. The α2 isoform of the Na+/K+-ATPase regulates intracellular Ca2+ signaling, contractility and pathological hypertrophy. The α3 isoform of the Na+/K+-ATPase may also be a target for cardiac hypertrophy. Restoration of cardiac Na+/K+-ATPase expression may be an effective approach for prevention of cardiac remodeling. In this article, we will overview: (1 the distribution and function of isoform specific Na+/K+-ATPase in the cardiomyocytes. (2 the role of cardiac Na+/K+-ATPase in the regulation of cell signaling, contractility, cardiac hypertrophy and fibrosis in vitro and in vivo. Selective targeting of cardiac Na+/K+-ATPase isoform may offer a new target for the prevention of cardiac remodeling.

Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

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Béziau, Delphine M; Toussaint, Fanny; Blanchette, Alexandre; Dayeh, Nour R; Charbel, Chimène; Tardif, Jean-Claude; Dupuis, Jocelyn; Ledoux, Jonathan

2015-01-01

Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the

Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

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Delphine M Béziau

Full Text Available Phospholipase C (PLC comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA, pulmonary (PA and middle cerebral arteries (MCA. mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA, δ4 (only expressed in MCA, η1 (expressed in all but MA and ζ (not detected in any vascular beds tested. The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1 in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found

Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

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Mirco Müller

Full Text Available Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms.

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Halling Linder, Cecilia; Narisawa, Sonoko; Millán, José Luis; Magnusson, Per

2009-11-01

Three circulating human bone alkaline phosphatase (BALP) isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue. The present study was designed to correlate differing glycosylation patterns of each BALP isoform with their catalytic activity towards presumptive physiological substrates and to compare those properties with two recombinant isoforms of the tissue-nonspecific ALP (TNALP) isozyme, i.e., TNALP-flag, used extensively for mutation analysis of hypophosphatasia mutations and sALP-FcD(10), a chimeric enzyme recently used as therapeutic drug in a mouse model of infantile hypophosphatasia. The BALP isoforms were prepared from human osteosarcoma (SaOS-2) cells and the kinetic properties were evaluated using the synthetic substrate p-nitrophenylphosphate (pNPP) at pH 7.4 and 9.8, and the three suggested endogenous physiological substrates, i.e., inorganic pyrophosphate (PP(i)), pyridoxal 5'-phosphate (PLP), and phosphoethanolamine (PEA) at pH 7.4. Qualitative glycosylation differences were also assessed by lectin binding and precipitation. The k(cat)/K(M) was higher for B2 for all the investigated substrates. The catalytic activity towards PEA was essentially undetectable. The kinetic activity for TNALP-flag and sALP-FcD(10) was similar to the activity of the human BALP isoforms. The BALP isoforms differed in their lectin binding properties and dose-dependent lectin precipitation, which also demonstrated differences between native and denatured BALP isoforms. The observed differences in lectin specificity were attributed to N-linked carbohydrates. In conclusion, we demonstrate significantly different catalytic properties among the BALP isoforms due to structural differences in posttranslational glycosylation. Our data also suggests that PEA is not an endogenous substrate for the BALP isoforms or for the recombinant TNALP isoforms. The TNALP

Oxygenation properties and isoform diversity of snake hemoglobins.

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Storz, Jay F; Natarajan, Chandrasekhar; Moriyama, Hideaki; Hoffmann, Federico G; Wang, Tobias; Fago, Angela; Malte, Hans; Overgaard, Johannes; Weber, Roy E

2015-11-01

Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer-dimer dissociation. However, standardized comparative data are lacking for snake Hbs, and the Hb isoform composition of snake red blood cells has not been systematically characterized. Here we present the results of an integrated analysis of snake Hbs and the underlying α- and β-type globin genes to characterize 1) Hb isoform composition of definitive erythrocytes, and 2) the oxygenation properties of isolated isoforms as well as composite hemolysates. We used species from three families as subjects for experimental studies of Hb function: South American rattlesnake, Crotalus durissus (Viperidae); Indian python, Python molurus (Pythonidae); and yellow-bellied sea snake, Pelamis platura (Elapidae). We analyzed allosteric properties of snake Hbs in terms of the Monod-Wyman-Changeux model and Adair four-step thermodynamic model. Hbs from each of the three species exhibited high intrinsic O2 affinities, low cooperativities, small Bohr factors in the absence of phosphates, and high sensitivities to ATP. Oxygenation properties of the snake Hbs could be explained entirely by allosteric transitions in the quaternary structure of intact tetramers, suggesting that ligation-dependent dissociation of Hb tetramers into αβ-dimers is not a universal feature of snake Hbs. Surprisingly, the major Hb isoform of the South American rattlesnake is homologous to the minor HbD of other amniotes and, contrary to the pattern of Hb isoform differentiation in birds and turtles, exhibits a lower O2 affinity than the HbA isoform. Copyright © 2015 the American Physiological Society.

Hemoglobin isoform differentiation and allosteric regulation of oxygen binding in the turtle, Trachemys scripta

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Damsgaard, Christian; Storz, Jay F.; Hoffmann, Federico G.

2013-01-01

When freshwater turtles acclimatize to winter hibernation, there is a gradual transition from aerobic to anaerobic metabolism, which may require adjustments of blood O2 transport before turtles become anoxic. Here, we report the effects of protons, anionic cofactors, and temperature on the O2......-binding properties of isolated hemoglobin (Hb) isoforms, HbA and HbD, in the turtle Trachemys scripta. We determined the primary structures of the constituent subunits of the two Hb isoforms, and we related the measured functional properties to differences in O2 affinity between untreated hemolysates from...... turtles that were acclimated to normoxia and anoxia. Our data show that HbD has a consistently higher O2 affinity compared with HbA, whereas Bohr and temperature effects, as well as thiol reactivity, are similar. Although sequence data show amino acid substitutions at two known β-chain ATP-binding site...

Specific nuclear localizing sequence directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner.

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Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

2012-01-01

Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

Biochemical characterization of individual human glycosylated pro-insulin-like growth factor (IGF)-II and big-IGF-II isoforms associated with cancer.

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Greenall, Sameer A; Bentley, John D; Pearce, Lesley A; Scoble, Judith A; Sparrow, Lindsay G; Bartone, Nicola A; Xiao, Xiaowen; Baxter, Robert C; Cosgrove, Leah J; Adams, Timothy E

2013-01-04

Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed "pro" and "big" IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling.

Biochemical Characterization of Individual Human Glycosylated pro-Insulin-like Growth Factor (IGF)-II and big-IGF-II Isoforms Associated with Cancer

Science.gov (United States)

Greenall, Sameer A.; Bentley, John D.; Pearce, Lesley A.; Scoble, Judith A.; Sparrow, Lindsay G.; Bartone, Nicola A.; Xiao, Xiaowen; Baxter, Robert C.; Cosgrove, Leah J.; Adams, Timothy E.

2013-01-01

Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed “pro” and “big” IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling. PMID:23166326

Isoform expression in the multiple soluble malate dehydrogenase of Hoplias malabaricus (Erythrinidae, Characiformes

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M. R. Aquino-Silva

Full Text Available Kinetic properties and thermal stabilities of Hoplias malabaricus liver and skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to further study the possible sMDH-A* locus duplication evolved from a recent tandem duplication. Both A (A1 and A2 and B isoforms had similar optima pH (7.5-8.0. While Hoplias A isoform could not be characterized as thermostable, B could as thermolabile. A isoforms differed from B isoform in having higher Km values for oxaloacetate. The possibly duplicated A2 isoform showed higher substrate affinity than the A1. Hoplias duplicated A isoforms may influence the direction of carbon flow between glycolisis and gluconeogenesis.

Isoform expression in the multiple soluble malate dehydrogenase of Hoplias malabaricus (Erythrinidae, Characiformes

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Aquino-Silva M. R.

2003-01-01

Full Text Available Kinetic properties and thermal stabilities of Hoplias malabaricus liver and skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to further study the possible sMDH-A* locus duplication evolved from a recent tandem duplication. Both A (A1 and A2 and B isoforms had similar optima pH (7.5-8.0. While Hoplias A isoform could not be characterized as thermostable, B could as thermolabile. A isoforms differed from B isoform in having higher Km values for oxaloacetate. The possibly duplicated A2 isoform showed higher substrate affinity than the A1. Hoplias duplicated A isoforms may influence the direction of carbon flow between glycolisis and gluconeogenesis.

Molecular characterization of elongase of very long-chain fatty acids 6 (elovl6) genes in Misgurnus anguillicaudatus and their potential roles in adaptation to cold temperature.

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Chen, Jingwen; Cui, Yun; Yan, Jie; Jiang, Jimin; Cao, Xiaojuan; Gao, Jian

2018-08-05

Elongase of very long-chain fatty acids 6 (ELOVL6) is a rate-limiting enzyme catalyzing elongation of saturated and monounsaturated long-chain fatty acid. Although functional characteristics of Elovl6 have been demonstrated in mammal, the role of elovl6 in fish remains unclear. In this study, we firstly cloned three isoforms of elovl6 (elovl6a, elovl6b and elovl6-like) from loach (Misgurnus anguillicaudatus). Molecular characterizations of the three elovl6 isoforms in loach and their expressions of early life stages and different tissues were then determined. We also functionally characterized the three elovl6 isoforms using heterologous expression in baker's yeast. Results obtained here showed the three elovl6 proteins in loach can elongate C16:0 and C16:1 to C18:0 and C18:1, respectively. At last, to confirm the role of three loach elovl6 isoforms for elongation of fatty acids in adaption to cold stress, differences in skin histological structures, body fatty acid compositions, expressions of four hepatic lipogenesis or lipolysis related genes, and expressions of the three elovl6 isoforms and their related gene uncoupling protein 1 (ucp1) in different tissues were investigated in the loach reared in two different water temperatures (28 °C and 4 °C) for ten days. Cold stress increased ratios of C18/C16 and C20:5n-3/C18:3n-3 in loach body, and induced expressions of hepatic acyl-CoA delta-9 desaturase 1 (scd1), sterol-regulator element-binding protein 1 (srebp1), carnitine palmitoyltransferase 1 (cpt1) and fatty acid synthase (fas). Meanwhile, significant differences were found in expressions of the three elovl6 isoforms in different tissues between 28 °C and 4 °C groups. Overall, this study suggests that the three elovl6 isoforms in loach have ability to elongate C16 to C18, and elovl6 proteins in loach may play a role in adaptation to cold stress. Copyright © 2018 Elsevier B.V. All rights reserved.

Isoform-selective regulation of glycogen phosphorylase by energy deprivation and phosphorylation in astrocytes

DEFF Research Database (Denmark)

Müller, Margit S; Pedersen, Sofie E; Walls, Anne B

2015-01-01

understood. In the present study, we used siRNA-mediated differential knockdown of the two isoforms of GP expressed in astrocytes, muscle isoform (GPMM), and brain isoform (GPBB), to analyze isoform-specific regulatory characteristics in a cellular setting. Subsequently, we tested the response of each...

Functional studies of sodium pump isoforms

DEFF Research Database (Denmark)

Clausen, Michael Jakob

The Na+,K+-ATPase is an essential ion pump found in all animal cells. It uses the energy from ATP hydrolysis to export three Na+ and import two K+, both against their chemical gradients and for Na+ also against the electrical potential. Mammals require four Na+,K+-ATPase isoforms that each have...... unique expression profiles and specialized functional features. We use a Two Electrode Voltage Clamp setup to determine pre-steady-state and steady-state characteristics of each isoform and design chimeras to pin-point the structural elements responsible for observed differences. With this strategy we...

Receptor-isoform-selective insulin analogues give tissue-preferential effects

DEFF Research Database (Denmark)

Vienberg, Sara Gry; Bouman, Stephan D; Sørensen, Heidi

2011-01-01

The relative expression patterns of the two IR (insulin receptor) isoforms, +/- exon 11 (IR-B/IR-A respectively), are tissue-dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms to test whether tissue-preferential biological effects can...... be attained. In rats and mice, IR-B is the most prominent isoform in the liver (> 95%) and fat (> 90%), whereas in muscles IR-A is the dominant isoform (> 95%). As a consequence, the insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency [compared with HI...... (human insulin)] for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, which has an increased affinity for human IR-B, had higher relative potencies (compared with HI...

The small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma and full-length FOXP1 exert similar oncogenic and transcriptional activity in human B cells.

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van Keimpema, Martine; Grüneberg, Leonie J; Schilder-Tol, Esther J M; Oud, Monique E C M; Beuling, Esther A; Hensbergen, Paul J; de Jong, Johann; Pals, Steven T; Spaargaren, Marcel

2017-03-01

The forkhead transcription factor FOXP1 is generally regarded as an oncogene in activated B cell-like diffuse large B-cell lymphoma. Previous studies have suggested that a small isoform of FOXP1 rather than full-length FOXP1, may possess this oncogenic activity. Corroborating those studies, we herein show that activated B cell-like diffuse large B-cell lymphoma cell lines and primary activated B cell-like diffuse large B-cell lymphoma cells predominantly express a small FOXP1 isoform, and that the 5'-end of the Foxp1 gene is a common insertion site in murine lymphomas in leukemia virus- and transposon-mediated insertional mutagenesis screens. By combined mass spectrometry, (quantative) reverse transcription polymerase chain reaction/sequencing, and small interfering ribonucleic acid-mediated gene silencing, we determined that the small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma lacks the N-terminal 100 amino acids of full-length FOXP1. Aberrant overexpression of this FOXP1 isoform (ΔN100) in primary human B cells revealed its oncogenic capacity; it repressed apoptosis and plasma cell differentiation. However, no difference in potency was found between this small FOXP1 isoform and full-length FOXP1. Furthermore, overexpression of full-length FOXP1 or this small FOXP1 isoform in primary B cells and diffuse large B-cell lymphoma cell lines resulted in similar gene regulation. Taken together, our data indicate that this small FOXP1 isoform and full-length FOXP1 have comparable oncogenic and transcriptional activity in human B cells, suggesting that aberrant expression or overexpression of FOXP1, irrespective of the specific isoform, contributes to lymphomagenesis. These novel insights further enhance the value of FOXP1 for the diagnostics, prognostics, and treatment of diffuse large B-cell lymphoma patients. Copyright© Ferrata Storti Foundation.

The C-terminal domain of the nuclear factor I-B2 isoform is glycosylated and transactivates the WAP gene in the JEG-3 cells

International Nuclear Information System (INIS)

Mukhopadhyay, Sudit S.; Rosen, Jeffrey M.

2007-01-01

The transcription factor nuclear factor I (NFI) has been shown previously both in vivo and in vitro to be involved in the cooperative regulation of whey acidic protein (WAP) gene transcription along with the glucocorticoid receptor and STAT5. In addition, one of the specific NFI isoforms, NFI-B2, was demonstrated in transient co-transfection experiments in JEG cells, which lack endogenous NFI, to be preferentially involved in the cooperative regulation of WAP gene expression. A comparison of the DNA-binding specificities of the different NFI isoforms only partially explained their differential ability to activate the WAP gene transcription. Here, we analyzed the transactivation regions of two NFI isoforms by making chimeric proteins between the NFI-A and B isoforms. Though, their DNA-binding specificities were not altered as compared to the corresponding wild-type transcription factors, the C-terminal region of the NFI-B isoform was shown to preferentially activate WAP gene transcription in cooperation with GR and STAT5 in transient co-transfection assays in JEG-3 cells. Furthermore, determination of serine and threonine-specific glycosylation (O-linked N-acetylglucosamine) of the C-terminus of the NFI-B isoform suggested that the secondary modification by O-GlcNAc might play a role in the cooperative regulation of WAP gene transcription by NFI-B2 and STAT5

Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72 isoforms

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Sehgal SA

2015-07-01

Full Text Available Sheikh Arslan Sehgal,1,2 Shazia Mannan,2,* Sumaira Kanwal,2,* Ishrat Naveed,1 Asif Mir1 1Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan; 2Department of Biosciences, COMSATS Institute of Information Technology, Sahiwal, Pakistan *These authors contributed equally to this work Abstract: Schizophrenia (SZ, a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with d-amino acid oxidase activator (DAOA, G72. Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor–ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ. Keywords: schizophrenia, bioinformatics, modeling, docking, DAOA, G72, DAO, computer-aided drug designing, phylogenetic analysis, d-amino acid oxidase

Learning-dependent gene expression of CREB1 isoforms in the molluscan brain

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Hisayo Sadamoto

2010-05-01

Full Text Available Cyclic AMP-responsive element binding protein1 (CREB1 has multiple functions in gene regulation. Various studies have reported that CREB1-dependent gene induction is necessary for memory formation and long-lasting behavioral changes in both vertebrates and invertebrates. In the present study, we characterized Lymnaea CREB1 (LymCREB1 mRNA isoforms of spliced variants in the central nervous system (CNS of the pond snail Lymnaea stagnalis. Among these spliced variants, the three isoforms that code a whole LymCREB1 protein are considered to be the activators for gene regulation. The other four isoforms, which code truncated LymCREB1 proteins with no kinase inducible domain, are the repressors. For a better understanding of the possible roles of different LymCREB1 isoforms, the expression level of these isoform mRNAs was investigated by a real-time quantitative RT-PCR method. Further, we examined the changes in gene expression for all the isoforms in the CNS after conditioned taste aversion (CTA learning or backward conditioning as a control. The results showed that CTA learning increased LymCREB1 gene expression, but it did not change the activator/repressor ratio. Our findings showed that the repressor isoforms, as well as the activator ones, are expressed in large amounts in the CNS, and the gene expression of CREB1 isoforms appeared to be specific for the given stimulus. This was the first quantitative analysis of the expression patterns of CREB1 isoforms at the mRNA level and their association with learning behavior.

Plectin isoforms as organizers of intermediate filament cytoarchitecture.

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Wiche, Gerhard; Winter, Lilli

2011-01-01

Intermediate filaments (IFs) form cytoplamic and nuclear networks that provide cells with mechanical strength. Perturbation of this structural support causes cell and tissue fragility and accounts for a number of human genetic diseases. In recent years, important additional roles, nonmechanical in nature, were ascribed to IFs, including regulation of signaling pathways that control survival and growth of the cells, and vectorial processes such as protein targeting in polarized cellular settings. The cytolinker protein plectin anchors IF networks to junctional complexes, the nuclear envelope and cytoplasmic organelles and it mediates their cross talk with the actin and tubulin cytoskeleton. These functions empower plectin to wield significant influence over IF network cytoarchitecture. Moreover, the unusual diversity of plectin isoforms with different N termini and a common IF-binding (C-terminal) domain enables these isoforms to specifically associate with and thereby bridge IF networks to distinct cellular structures. Here we review the evidence for IF cytoarchitecture being controlled by specific plectin isoforms in different cell systems, including fibroblasts, endothelial cells, lens fibers, lymphocytes, myocytes, keratinocytes, neurons and astrocytes, and discuss what impact the absence of these isoforms has on IF cytoarchitecture-dependent cellular functions.

Molecular Pharmacology of VEGF-A Isoforms: Binding and Signalling at VEGFR2.

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Peach, Chloe J; Mignone, Viviane W; Arruda, Maria Augusta; Alcobia, Diana C; Hill, Stephen J; Kilpatrick, Laura E; Woolard, Jeanette

2018-04-23

Vascular endothelial growth factor-A (VEGF-A) is a key mediator of angiogenesis, signalling via the class IV tyrosine kinase receptor family of VEGF Receptors (VEGFRs). Although VEGF-A ligands bind to both VEGFR1 and VEGFR2, they primarily signal via VEGFR2 leading to endothelial cell proliferation, survival, migration and vascular permeability. Distinct VEGF-A isoforms result from alternative splicing of the Vegfa gene at exon 8, resulting in VEGF xxx a or VEGF xxx b isoforms. Alternative splicing events at exons 5⁻7, in addition to recently identified posttranslational read-through events, produce VEGF-A isoforms that differ in their bioavailability and interaction with the co-receptor Neuropilin-1. This review explores the molecular pharmacology of VEGF-A isoforms at VEGFR2 in respect to ligand binding and downstream signalling. To understand how VEGF-A isoforms have distinct signalling despite similar affinities for VEGFR2, this review re-evaluates the typical classification of these isoforms relative to the prototypical, “pro-angiogenic” VEGF 165 a. We also examine the molecular mechanisms underpinning the regulation of VEGF-A isoform signalling and the importance of interactions with other membrane and extracellular matrix proteins. As approved therapeutics targeting the VEGF-A/VEGFR signalling axis largely lack long-term efficacy, understanding these isoform-specific mechanisms could aid future drug discovery efforts targeting VEGF receptor pharmacology.

Comprehensive analysis of tropomyosin isoforms in skeletal muscles by top-down proteomics.

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Jin, Yutong; Peng, Ying; Lin, Ziqing; Chen, Yi-Chen; Wei, Liming; Hacker, Timothy A; Larsson, Lars; Ge, Ying

2016-04-01

Mammalian skeletal muscles are heterogeneous in nature and are capable of performing various functions. Tropomyosin (Tpm) is a major component of the thin filament in skeletal muscles and plays an important role in controlling muscle contraction and relaxation. Tpm is known to consist of multiple isoforms resulting from different encoding genes and alternative splicing, along with post-translational modifications. However, a systematic characterization of Tpm isoforms in skeletal muscles is still lacking. Therefore, we employed top-down mass spectrometry (MS) to identify and characterize Tpm isoforms present in different skeletal muscles from multiple species, including swine, rat, and human. Our study revealed that Tpm1.1 and Tpm2.2 are the two major Tpm isoforms in swine and rat skeletal muscles, whereas Tpm1.1, Tpm2.2, and Tpm3.12 are present in human skeletal muscles. Tandem MS was utilized to identify the sequences of the major Tpm isoforms. Furthermore, quantitative analysis revealed muscle-type specific differences in the abundance of un-modified and modified Tpm isoforms in rat and human skeletal muscles. This study represents the first systematic investigation of Tpm isoforms in skeletal muscles, which not only demonstrates the capabilities of top-down MS for the comprehensive characterization of skeletal myofilament proteins but also provides the basis for further studies on these Tpm isoforms in muscle-related diseases.

Differential Signature of the Centrosomal MARK4 Isoforms in Glioma

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Ivana Magnani

2011-01-01

Full Text Available Background: MAP/microtubule affinity-regulating kinase 4 (MARK4 is a serine-threonine kinase expressed in two spliced isoforms, MARK4L and MARK4S, of which MARK4L is a candidate for a role in neoplastic transformation. Methods: We performed mutation analysis to identify sequence alterations possibly affecting MARK4 expression. We then investigated the MARK4L and MARK4S expression profile in 21 glioma cell lines and 36 tissues of different malignancy grades, glioblastoma-derived cancer stem cells (GBM CSCs and mouse neural stem cells (NSCs by real-time PCR, immunoblotting and immunohistochemistry. We also analyzed the sub-cellular localisation of MARK4 isoforms in glioma and normal cell lines by immunofluorescence. Results: Mutation analysis rules out sequence variations as the cause of the altered MARK4 expression in glioma. Expression profiling confirms that MARK4L is the predominant isoform, whereas MARK4S levels are significantly decreased in comparison and show an inverse correlation with tumour grade. A high MARK4L/MARK4S ratio also characterizes undifferentiated cells, such as GBM CSCs and NSCs. Accordingly, only MARK4L is expressed in brain neurogenic regions. Moreover, while both MARK4 isoforms are localised to the centrosome and midbody in glioma and normal cells, the L isoform exhibits an additional nucleolar localisation in tumour cells. Conclusions: The observed switch towards MARK4L suggests that the balance between the MARK4 isoforms is carefully guarded during neural differentiation but may be subverted in gliomagenesis. Moreover, the MARK4L nucleolar localisation in tumour cells features this MARK4 isoform as a nucleolus-associated tumour marker.

Developmental changes in circulating IL-8/CXCL8 isoforms in neonates.

Science.gov (United States)

Maheshwari, Akhil; Voitenok, Nikolai N; Akalovich, Svetlana; Shaik, Sadiq S; Randolph, David A; Sims, Brian; Patel, Rakesh P; Killingsworth, Cheryl R; Fallon, Michael B; Ohls, Robin K

2009-04-01

Interleukin-8 (IL-8/CXCL8) is widely expressed in fetal tissues although inflammatory changes are not seen. Circulating IL-8 is comprised of an endothelial-derived [ala-IL-8](77) isoform and another, more potent [ser-IL-8](72) secreted by most other cells; [ala-IL-8](77) can be converted into [ser-IL-8](72) by proteolytic removal of an N-terminal pentapeptide from [ala-IL-8](77). In this study, we show [ala-IL-8](77) is the predominant circulating isoform of IL-8 in premature neonates but not in term neonates/adults, who have [ser-IL-8](72) as the major isoform. This isoform switch from the less potent [ala-IL-8](77) to [ser-IL-8](72) correlates with a maturational increase in the neutrophil chemotactic potency of plasma IL-8. The emergence of [ser-IL-8](72) as the major isoform is likely due to increased plasma [ala-IL-8](77)-convertase activity and/or changes in the cellular sources of IL-8. Developmental changes in IL-8 isoforms may serve to minimize its inflammatory effects in the fetus and also provide a mechanism to restore its full activity after birth.

Response of slow and fast muscle to hypothyroidism: maximal shortening velocity and myosin isoforms

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Caiozzo, V. J.; Herrick, R. E.; Baldwin, K. M.

1992-01-01

This study examined both the shortening velocity and myosin isoform distribution of slow- (soleus) and fast-twitch (plantaris) skeletal muscles under hypothyroid conditions. Adult female Sprague-Dawley rats were randomly assigned to one of two groups: control (n = 7) or hypothyroid (n = 7). In both muscles, the relative contents of native slow myosin (SM) and type I myosin heavy chain (MHC) increased in response to the hypothyroid treatment. The effects were such that the hypothyroid soleus muscle expressed only the native SM and type I MHC isoforms while repressing native intermediate myosin and type IIA MHC. In the plantaris, the relative content of native SM and type I MHC isoforms increased from 5 to 13% and from 4 to 10% of the total myosin pool, respectively. Maximal shortening velocity of the soleus and plantaris as measured by the slack test decreased by 32 and 19%, respectively, in response to hypothyroidism. In contrast, maximal shortening velocity as estimated by force-velocity data decreased only in the soleus (-19%). No significant change was observed for the plantaris.

VEGF111b, a new member of VEGFxxxb isoforms and induced by mitomycin C, inhibits angiogenesis

Energy Technology Data Exchange (ETDEWEB)

Gu, Fang; Li, Xiuli [Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing (China); Kong, Jian [Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing (China); Pan, Bing [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Sun, Min [Department of Obstetrics and Gynecology, Tangdu Hospital, Fourth Military Medical University, Xian (China); Zheng, Lemin, E-mail: zhengl@bjmu.edu.cn [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Yao, Yuanqing, E-mail: yqyao@126.com [Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing (China)

2013-11-08

Highlights: •We discovered a new member of VEGFxxxb family-VEGF111b. •We found VEGF111b mRNA and protein can be induced by mitomycin C. •We confirmed VEGF111b over-expression inhibits angiogenesis. •VEGF111b inhibits angiogenesis through inhibiting VEGF-R2/PI3K/Akt and VEGF-R2/ERK1/2 phosphorylation. -- Abstract: Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA{sub 3.1} empty vector, pcDNA{sub 3.1}-VEGF111b or pcDNA{sub 3.1}-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.

VEGF111b, a new member of VEGFxxxb isoforms and induced by mitomycin C, inhibits angiogenesis

International Nuclear Information System (INIS)

Gu, Fang; Li, Xiuli; Kong, Jian; Pan, Bing; Sun, Min; Zheng, Lemin; Yao, Yuanqing

2013-01-01

Highlights: •We discovered a new member of VEGFxxxb family-VEGF111b. •We found VEGF111b mRNA and protein can be induced by mitomycin C. •We confirmed VEGF111b over-expression inhibits angiogenesis. •VEGF111b inhibits angiogenesis through inhibiting VEGF-R2/PI3K/Akt and VEGF-R2/ERK1/2 phosphorylation. -- Abstract: Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA 3.1 empty vector, pcDNA 3.1 -VEGF111b or pcDNA 3.1 -VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth

Expression of various sarcomeric tropomyosin isoforms in equine striated muscles

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Syamalima Dube

2017-06-01

Full Text Available In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin. Tropomyosin (TPM, a coiled-coil dimeric protein, is a component of thin filament in striated muscles. In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4 generate a multitude of TPM isoforms via alternate splicing and/or using different promoters. Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited. Hence, we undertook a comprehensive exploratory study of various TPM isoforms from horse heart and skeletal muscle. We have cloned and sequenced two sarcomeric isoforms of the TPM1 gene called TPM1α and TPM1κ, one sarcomeric isoform of the TPM2 and one of the TPM3 gene, TPM2α and TPM3α respectively. By qRT-PCR using both relative expression and copy number, we have shown that TPM1α expression compared to TPM1κ is very high in heart. On the other hand, the expression of TPM1α is higher in skeletal muscle compared to heart. Further, the expression of TPM2α and TPM3α are higher in skeletal muscle compared to heart. Using western blot analyses with CH1 monoclonal antibody we have shown the high expression levels of sarcomeric TPM proteins in cardiac and skeletal muscle. Due to the paucity of isoform specific antibodies we cannot specifically detect the expression of TPM1κ in horse striated muscle. To the best of our knowledge this is the very first report on the characterization of sarcmeric TPMs in horse striated muscle.

Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle [version 1; referees: 2 approved, 1 approved with reservations

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Christine Chaponnier

2016-03-01

Full Text Available Higher vertebrates express six different highly conserved actin isoforms that can be classified in three subgroups: 1 sarcomeric actins, α-skeletal (α-SKA and α-cardiac (α-CAA, 2 smooth muscle actins (SMAs, α-SMA and γ-SMA, and 3 cytoplasmic actins (CYAs, β-CYA and γ-CYA. The variations among isoactins, in each subgroup, are due to 3-4 amino acid differences located in their acetylated N-decapeptide sequence. The first monoclonal antibody (mAb against an actin isoform (α-SMA was produced and characterized in our laboratory in 1986 (Skalli et al., 1986. We have further obtained mAbs against the 5 other isoforms. In this report, we focus on the mAb anti-α-SKA and anti-α-CAA obtained after immunization of mice with the respective acetylated N-terminal decapeptides using the Repetitive Immunizations at Multiple Sites Strategy (RIMMS. In addition to the identification of their epitope by immunoblotting, we describe the expression of the 2 sarcomeric actins in mature skeletal muscle and during muscle repair after micro-lesions. In particular, we analyze the expression of α-CAA, α-SKA and α-SMA by co-immunostaining in a time course frame during the muscle repair process. Our results indicate that a restricted myocyte population expresses α-CAA and suggest a high capacity of self-renewal in muscle cells. These antibodies may represent a helpful tool for the follow-up of muscle regeneration and pathological changes.

VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

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Gareth W. Fearnley

2016-05-01

Full Text Available Vascular endothelial growth factor A (VEGF-A binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145 promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes.

VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis.

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Fearnley, Gareth W; Smith, Gina A; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T; Zachary, Ian C; Tomlinson, Darren C; Harrison, Michael A; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

2016-05-15

Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A-VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor-ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. © 2016. Published by The Company of Biologists Ltd.

Induction of Shikimic Acid Pathway Enzymes by Light in Suspension Cultured Cells of Parsley (Petroselinum crispum) 1

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McCue, Kent F.; Conn, Eric E.

1990-01-01

Light treatment of suspension cultured cells of parsley (Petroselinum crispum) was shown to increase the activity of the shikimic acid pathway enzyme, 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase (EC 4.1.2.15). DAHP synthase activity was assayed for two isoforms, DS-Mn and DS-Co (RJ Ganson, TA d'Amato, RA Jensen [1986] Plant Physiol 82: 203-210). Light increased the enzymatic activity of the plastidic isoform DS-Mn as much as 2-fold, averaging 1.6-fold with >95% confidence. The cytosolic isoform DS-Co was unaffected. Cycloheximide and actinomycin D, translational and transcriptional inhibitors, respectively, both reversed induction of DS-Mn by light suggesting transcriptional regulation of the gene. Chorismate mutase activity was assayed for the two isoforms CM I and CM II (BK Singh, JA Connelly, EE Conn [1985] Arch Biochem Biophys 243: 374-384). Treatment by light did not significantly affect either chorismate mutase isoform. The ratio of the two chorismate mutase isoforms changed during the growth cycle, with an increase in the ratio of plastidic to cytosolic isoforms occurring towards the end of logarithmic growth. PMID:16667741

Eff ect of vitamin E isoforms on the primary intention Eff ect of vitamin E isoforms on the primary intention skin wound healing of diabetic rats

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Bijo Elsy

2017-10-01

Full Text Available Introduction: Impaired wound healing events is a common complication in diabetes. One of the effective nutritional antioxidant on skin wound healing is vitamin E which contains saturated tocopherol and unsaturated tocotrienol forms. This present study is designed to explore the effect of different vitamin E isoforms on stitched skin wound in both healthy and diabetic rats. Materials and Methods: Forty eight albino rats were divided into eight groups; healthy control, diabetic control, healthy treated (d-α-tocopherol, d-δ-TRF and co-administrated and diabetic treated (d-αtocopherol, d-δ-TRF and co-administrated. Diabetes was induced through single subcutaneous injection of alloxan at the dose of 100 mg/kg. Treated groups were administered d-a-tocopherol (200 mg/kg, d-δ-TRF (200 mg/kg and co-administration (100 mg/kg of these two compounds each orally and daily for three weeks. A horizontal skin incision was made on right mid-thigh region at 2.95 ± 0.17cm in length and wound was closed with an absorbable suture. Results: Histopathological and histomorphological results at the end of 3rd week revealed that the d-δ-TRF treated groups promote the regeneration and reorganization of epidermal and dermal components in healing of primary intention more effectively than the d-α-tocopherol and co-administrated groups. Conclusion: It is concluded that among different vitamin E isoforms the d-δ-TRF appears to be a more effective nutritional antioxidant on skin wound healing in both healthy and diabetics.

Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis

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Jifeng Zhang

2015-01-01

Full Text Available Endophilin isoforms perform distinct characteristics in their interactions with N-type Ca2+ channels and dynamin. However, precise functional differences for the endophilin isoforms on synaptic vesicle (SV endocytosis remain unknown. By coupling RNA interference and electrophysiological recording techniques in cultured rat hippocampal neurons, we investigated the functional differences of three isoforms of endophilin in SV endocytosis. The results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations. Similar results were found using endophilin2 knockdown neurons, whereas endophilin3 siRNA exhibited no change compared with control neurons. Endophilin1 and endophilin2 affected SV endocytosis, but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone. This result suggested that endophilin1 and endophilin2 functioned together but not independently during SV endocytosis. Taken together, our results indicate that SV endocytosis is sustained by endophilin1 and endophilin2 isoforms, but not by endophilin3, in primary cultured hippocampal neurons.

Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72) isoforms.

Science.gov (United States)

Sehgal, Sheikh Arslan; Mannan, Shazia; Kanwal, Sumaira; Naveed, Ishrat; Mir, Asif

2015-01-01

Schizophrenia (SZ), a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with D-amino acid oxidase activator (DAOA, G72). Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor-ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ.

The Role of Akt Isoforms in Colorectal Cancer

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2015-09-01

AD_________________ Award Number: W81XWH-13-1-0198 TITLE: The Role of Akt Isoforms in Colorectal Cancer PRINCIPAL INVESTIGATOR: Jatin Roper...CONTRACT NUMBER The Role of Akt Isoforms in Colorectal Cancer 5b. GRANT NUMBER W81XWH-13-1-0198 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER...substantially reduces colorectal tumorigenesis in our genetically engineered mouse model. We also successfully ablated novel downstream targets of Akt in our

Altered α-synuclein, parkin, and synphilin isoform levels in multiple system atrophy brains

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Brudek, Tomasz; Winge, Kristian; Rasmussen, Nadja Bredo

2016-01-01

Together with Parkinson's disease (PD) and dementia with Lewy bodies, multiple system atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α-synucleinopathies. Previously, it has been shown that α-synuclein, parkin, and synphilin-1 display disease-specific transcript......Together with Parkinson's disease (PD) and dementia with Lewy bodies, multiple system atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α-synucleinopathies. Previously, it has been shown that α-synuclein, parkin, and synphilin-1 display disease......-specific transcription patterns in frontal cortex in PD, dementia with Lewy bodies, and MSA, and thus may mediate the development of α-synucleinopathies. In this study, the differential expression of α-synuclein isoforms on transcriptional and translational levels was ascertained in MSA patients in comparison with PD......-synuclein in the brain. We report differential expression of α-synuclein, parkin, and synphilin-1 isoforms in multiple system atrophy (MSA) versus Parkinson's disease and normal control brains. We have focused on brain regions that are severely affected by α-synuclein pathology and neurodegeneration in MSA. The reported...

Molecular cloning and characterization of a novel isoform of the non-canonical poly(A) polymerase PAPD7

Energy Technology Data Exchange (ETDEWEB)

Ogami, Koichi; Cho, Rihe [Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Hoshino, Shin-ichi, E-mail: hoshino@phar.nagoya-cu.ac.jp [Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan)

2013-03-01

Highlights: ► So far, only an enzymatically inactive isoform of PAPD7 was reported. ► The novel isoform: PAPD7 l shows robust nucleotidyl transferase activity. ► The newly identified amino terminal region is required for the activity. ► PAPD7 l localizes to the nucleoplasm. ► The N terminal region identified is also required for the nuclear localization. - Abstract: Non-canonical poly(A) polymerases (ncPAPs) catalyze the addition of poly(A) tail to the 3′ end of RNA to play pivotal roles in the regulation of gene expression and also in quality control. Here we identified a novel isoform of the 7th member of ncPAPs: PAPD7 (PAPD7 l), which contains 230 extra amino acids at the amino terminus of the previously identified PAPD7 (PAPD7 s). In sharp contrast to the inactive PAPD7 s, PAPD7 l showed robust nucleotidyl transferase activity when tethered to an RNA. A region required for the activity was localized to 187–219 aa, and this region was also required for the nuclear retention of PAPD7 l. Western blot analysis revealed that 94 kDa band (corresponding to PAPD7 l) but not 62 kDa band (corresponding to PAPD7 s) detected by PAPD7 antibody was specifically depleted by treatment with PAPD7 siRNA in both HeLa and U2OS cells. These results suggest that PAPD7 l is the major and active isoform of PAPD7 expressed in cells.

Effects of Andrographis paniculata and Orthosiphon stamineus extracts on the glucuronidation of 4-methylumbelliferone in human UGT isoforms.

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Ismail, Sabariah; Hanapi, Nur Aziah; Ab Halim, Mohd Rohaimi; Uchaipichat, Verawan; Mackenzie, Peter I

2010-05-14

The effects of Andrographis paniculata and Orthosiphon stamineus extracts on the in vitro glucuronidation of 4-methylumbelliferone (4MU) by recombinant human UGTs, UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7 and UGT2B15 were determined. The potential inhibitory effects of both of the extracts on the activity of each of the UGT isoforms were investigated using 4MU as the substrate. Incubations contained UDP-glucuronic acid (UDPGA) as the cofactor, MgCl(2), cell lysate of respective isoform, and 4MU at the approximate apparent K(m) or S(50) value of each isoform. Final concentrations of Andrographis paniculata and Orthosiphon stamineus extracts used were 0.025, 0.25, 2.5, 25 and 50 microg/mL and 0.01, 0.10, 1.0, 10 and 50 microg/mL respectively. Both extracts variably inhibited the activity of most of the isoforms in a concentration dependent manner. Andrographis paniculata extract was the better inhibitor of all the isoforms studied (IC(50) 1.70 microg/mL for UGT1A3, 2.57 microg/mL for UGT1A8, 2.82 microg/mL for UGT2B7, 5.00 micorg/mL for UGT1A1, 5.66 microg/mL for UGT1A6, 9.88 microg/mL for UGT1A7 and 15.66 microg/mL for UGT1A10). Both extracts showed less than 70% inhibition of UGT2B15, so the IC(50) values were >50 microg/mL. The inhibition of human UGTs by Andrographis paniculata and Orthosiphon stamineus extracts in vitro suggests a potential for drug-herbal extract interactions in the therapeutic setting.

Functional groups and reactivity of size-fractionated Aldrich humic acid

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Tanaka, Tadao, E-mail: tanaka.tadao26@jaea.go.jp [Nuclear Safety Research Center, Japan Atomic Energy Agency, Shirakata 2-4, Tokai, Naka, Ibaraki, 319-1195 (Japan)

2012-03-20

The complexation affinity of the Aldrich Chemicals humic acid with {sup 60}Co was examined with respect to molecular size of humic acid. Correlations between the affinity and the structures of humic acid were studied. At low humic acid concentration range, {sup 60}Co was interacted with the humic acid of size fraction over 100k Da (HA(100<)). With increasing humic acid concentration, the {sup 60}Co was preferentially interacted with the 30k-100k Da of humic acid (HA(30-100)). Fractionated HA(100<) and HA(30-100) were characterized from their FTIR (Fourier Transform Infra-Red), {sup 13}C NMR spectral analyses and acid-base titration curves. The HA(10<) having aliphatic branched structure, smaller amount of COOH group and large proton exchangeable capacity, seem to show low covalent bonding nature and high ion exchangeability in the complexation. In addition, steric hindrance may affect on the complexation, by winding up like random coils from the branched structure. The HA(30-100) is dominated with the aromatic COOH group and OH group and it may preferentially coordinate to {sup 60}Co by covalent binding. These presumptions were supported by XPS analysis, in which the biding energy of cobalt-humates was discussed.

Network-Based Isoform Quantification with RNA-Seq Data for Cancer Transcriptome Analysis.

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Wei Zhang

2015-12-01

Full Text Available High-throughput mRNA sequencing (RNA-Seq is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA, the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/.

NHS-A isoform of the NHS gene is a novel interactor of ZO-1.

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Sharma, Shiwani; Koh, Katrina S Y; Collin, Caitlin; Dave, Alpana; McMellon, Amy; Sugiyama, Yuki; McAvoy, John W; Voss, Anne K; Gécz, Jozef; Craig, Jamie E

2009-08-15

Mutations in the NHS (Nance-Horan Syndrome) gene lead to severe congenital cataracts, dental defects and sometimes mental retardation. NHS encodes two protein isoforms, NHS-A and -1A that display cell-type dependent differential expression and localization. Here we demonstrate that of these two isoforms, the NHS-A isoform associates with the cell membrane in the presence of intercellular contacts and it immunoprecipitates with the tight junction protein ZO-1 in MDCK (Madin Darby Canine Kidney) epithelial cells and in neonatal rat lens. The NHS-1A isoform however is a cytoplasmic protein. Both Nhs isoforms are expressed during mouse development. Immunolabelling of developing mouse with the anti-NHS antibody that detects both isoforms revealed the protein in the developing head including the eye and brain. It was primarily expressed in epithelium including neural epithelium and certain vascular endothelium but only weakly expressed in mesenchymal cells. In the epithelium and vascular endothelium the protein associated with the cell membrane and co-localized with ZO-1, which indirectly indicates expression of the Nhs-A isoform in these structures. Membrane localization of the protein in the lens vesicle similarly supports Nhs-A expression. In conclusion, the NHS-A isoform of NHS is a novel interactor of ZO-1 and may have a role at tight junctions. This isoform is important in mammalian development especially of the organs in the head.

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription.

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Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien; Pira, Dorcas; Angrand, Pierre-Olivier

2012-05-01

Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

RAGE receptor and its soluble isoforms in diabetes mellitus complications

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Mauren Isfer Anghebem Oliveira

2013-04-01

Full Text Available Chronic hyperglycemia, which is present in all types of diabetes, increases the formation of advanced glycation end-products (AGEs. The interaction of AGEs with receptor of advanced glycation end-products (RAGE initiates a cascade of pro-inflammatory and pro-coagulant processes that result in oxidative stress, stimulating the formation and accumulation of more AGE molecules. This cyclic process, denominated metabolic memory, may explain the persistency of diabetic vascular complications in patients with satisfactory glycemic control. The RAGE found in several cell membranes is also present in soluble isoforms (esRAGE and cRAGE, which are generated by alternative deoxyribonucleic acid splicing or by proteolytic cleavage. This review focuses on new research into these mediators as potential biomarkers for vascular complications in diabetes.

Proteogenomic Analysis Identifies a Novel Human SHANK3 Isoform

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Fahad Benthani

2015-05-01

Full Text Available Mutations of the SHANK3 gene have been associated with autism spectrum disorder. Individuals harboring different SHANK3 mutations display considerable heterogeneity in their cognitive impairment, likely due to the high SHANK3 transcriptional diversity. In this study, we report a novel interaction between the Mutated in colorectal cancer (MCC protein and a newly identified SHANK3 protein isoform in human colon cancer cells and mouse brain tissue. Hence, our proteogenomic analysis identifies a new human long isoform of the key synaptic protein SHANK3 that was not predicted by the human reference genome. Taken together, our findings describe a potential new role for MCC in neurons, a new human SHANK3 long isoform and, importantly, highlight the use of proteomic data towards the re-annotation of GC-rich genomic regions.

The human CD8β M-4 isoform dominant in effector memory T cells has distinct cytoplasmic motifs that confer unique properties.

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Deepshi Thakral

Full Text Available The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4 that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.

An abnormally glycosylated isoform of erythropoietin in hemangioblastoma is associated with polycythemia.

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Delanghe, Sigurd E; Dierick, Jan; Maenhout, Thomas M; Zabeau, Lennart; Tavernier, Jan; Claes, Kathleen; Bleyen, Joris; Delanghe, Joris R

2015-01-01

Hemangioblastomas express erythropoietin and the patients often present with polycythemia. Serum erythropoietin was measured using a commercial immunoassay, a functional erythropoietin assay and iso-electric focusing. Despite the polycythemia, serum erythropoietin remained low, while a functional erythropoietin-assay showed a 4-5 higher activity in serum compared to the immunoassay. Iso-electric focusing of serum erythropoietin indicated overrepresentation of highly sialylated erythropoietin isoforms produced by the tumor. As a result, altered affinity of the monoclonal antibody used in the immunoassay for the hypersialylated isoforms was suggested. Analysis of erythropoietin isoforms may be helpful in distinguishing the ectopic erythropoietin isoforms from normally glycosylated erythropoietin. Copyright © 2014 Elsevier B.V. All rights reserved.

The combined expression patterns of Ikaros isoforms characterize different hematological tumor subtypes.

Science.gov (United States)

Orozco, Carlos A; Acevedo, Andrés; Cortina, Lazaro; Cuellar, Gina E; Duarte, Mónica; Martín, Liliana; Mesa, Néstor M; Muñoz, Javier; Portilla, Carlos A; Quijano, Sandra M; Quintero, Guillermo; Rodriguez, Miriam; Saavedra, Carlos E; Groot, Helena; Torres, María M; López-Segura, Valeriano

2013-01-01

A variety of genetic alterations are considered hallmarks of cancer development and progression. The Ikaros gene family, encoding for key transcription factors in hematopoietic development, provides several examples as genetic defects in these genes are associated with the development of different types of leukemia. However, the complex patterns of expression of isoforms in Ikaros family genes has prevented their use as clinical markers. In this study, we propose the use of the expression profiles of the Ikaros isoforms to classify various hematological tumor diseases. We have standardized a quantitative PCR protocol to estimate the expression levels of the Ikaros gene exons. Our analysis reveals that these levels are associated with specific types of leukemia and we have found differences in the levels of expression relative to five interexonic Ikaros regions for all diseases studied. In conclusion, our method has allowed us to precisely discriminate between B-ALL, CLL and MM cases. Differences between the groups of lymphoid and myeloid pathologies were also identified in the same way.

Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

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Heger Christopher D

2010-12-01

Full Text Available Abstract Background Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. Methods Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. Results We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76% of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72% and 27% had only low levels of expression. Conclusions Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in

Revealing the functions of the transketolase enzyme isoforms in Rhodopseudomonas palustris using a systems biology approach.

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Chia-Wei Hu

Full Text Available BACKGROUND: Rhodopseudomonas palustris (R. palustris is a purple non-sulfur anoxygenic phototrophic bacterium that belongs to the class of proteobacteria. It is capable of absorbing atmospheric carbon dioxide and converting it to biomass via the process of photosynthesis and the Calvin-Benson-Bassham (CBB cycle. Transketolase is a key enzyme involved in the CBB cycle. Here, we reveal the functions of transketolase isoforms I and II in R. palustris using a systems biology approach. METHODOLOGY/PRINCIPAL FINDINGS: By measuring growth ability, we found that transketolase could enhance the autotrophic growth and biomass production of R. palustris. Microarray and real-time quantitative PCR revealed that transketolase isoforms I and II were involved in different carbon metabolic pathways. In addition, immunogold staining demonstrated that the two transketolase isoforms had different spatial localizations: transketolase I was primarily associated with the intracytoplasmic membrane (ICM but transketolase II was mostly distributed in the cytoplasm. Comparative proteomic analysis and network construction of transketolase over-expression and negative control (NC strains revealed that protein folding, transcriptional regulation, amino acid transport and CBB cycle-associated carbon metabolism were enriched in the transketolase I over-expressed strain. In contrast, ATP synthesis, carbohydrate transport, glycolysis-associated carbon metabolism and CBB cycle-associated carbon metabolism were enriched in the transketolase II over-expressed strain. Furthermore, ATP synthesis assays showed a significant increase in ATP synthesis in the transketolase II over-expressed strain. A PEPCK activity assay showed that PEPCK activity was higher in transketolase over-expressed strains than in the negative control strain. CONCLUSIONS/SIGNIFICANCE: Taken together, our results indicate that the two isoforms of transketolase in R. palustris could affect photoautotrophic growth

High Molecular Weight Isoforms of Growth Hormone In Cells of the Immune System

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Weigent, Douglas A.

2013-01-01

A substantial body of research exists to support the idea that cells of the immune system produce growth hormone (GH). However, the structure and mechanism of action of lymphocyte-derived GH continues to remain largely unknown. Here we present the results of Western analysis of whole cell extracts showing that different molecular weight isoforms of GH of approximately 100 kDa, 65 kDa, and 48 kDa can be detected in primary mouse cells of the immune system and in the mouse EL4 cell line. The identity of the 65 kDa and 48 kDa isoforms of GH were confirmed by mass spectrometry. The various isoforms were detected in both enriched T and B spleen cell populations. The large molecular weight isoform appears to reside primarily in the cytoplasm whereas the lower molecular weight 65 kDa and 48 kDa isoforms were detected primarily in the nucleus. These results also suggest that GH isoforms are induced by oxidative stress. In EL4 cells overexpressing GH, the expression of luciferase controlled by a promoter containing the antioxidant response element is increased almost three-fold above control. The data suggest that the induction of isoforms of the GH molecule in cells of the immune system may be an important mechanism of adaptation and/or protection of lymphoid cells under conditions of oxidative stress. PMID:21741628

Development of isoform-specific sensors of polypeptide GalNAc-transferase activity

DEFF Research Database (Denmark)

Song, Lina; Bachert, Collin; Schjoldager, Katrine T

2014-01-01

sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable......Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms....... Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal...

Evolution of the cAMP-dependent protein kinase (PKA catalytic subunit isoforms.

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Kristoffer Søberg

Full Text Available The 3',5'-cyclic adenosine monophosphate (cAMP-dependent protein kinase, or protein kinase A (PKA, pathway is one of the most versatile and best studied signaling pathways in eukaryotic cells. The two paralogous PKA catalytic subunits Cα and Cβ, encoded by the genes PRKACA and PRKACB, respectively, are among the best understood model kinases in signal transduction research. In this work, we explore and elucidate the evolution of the alternative 5' exons and the splicing pattern giving rise to the numerous PKA catalytic subunit isoforms. In addition to the universally conserved Cα1/Cβ1 isoforms, we find kinase variants with short N-termini in all main vertebrate classes, including the sperm-specific Cα2 isoform found to be conserved in all mammals. We also describe, for the first time, a PKA Cα isoform with a long N-terminus, paralogous to the PKA Cβ2 N-terminus. An analysis of isoform-specific variation highlights residues and motifs that are likely to be of functional importance.

Novel frataxin isoforms may contribute to the pathological mechanism of Friedreich ataxia.

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Haiyan Xia

Full Text Available Friedreich ataxia (FRDA is an inherited neurodegenerative disease caused by frataxin (FXN deficiency. The nervous system and heart are the most severely affected tissues. However, highly mitochondria-dependent tissues, such as kidney and liver, are not obviously affected, although the abundance of FXN is normally high in these tissues. In this study we have revealed two novel FXN isoforms (II and III, which are specifically expressed in affected cerebellum and heart tissues, respectively, and are functional in vitro and in vivo. Increasing the abundance of the heart-specific isoform III significantly increased the mitochondrial aconitase activity, while over-expression of the cerebellum-specific isoform II protected against oxidative damage of Fe-S cluster-containing aconitase. Further, we observed that the protein level of isoform III decreased in FRDA patient heart, while the mRNA level of isoform II decreased more in FRDA patient cerebellum compared to total FXN mRNA. Our novel findings are highly relevant to understanding the mechanism of tissue-specific pathology in FRDA.

C/EBPβ Isoforms Expression in the Rat Brain during the Estrous Cycle

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Valeria Hansberg-Pastor

2015-01-01

Full Text Available The CCAAT/enhancer-binding protein beta (C/EBPβ is a transcription factor expressed in different areas of the brain that regulates the expression of several genes involved in cell differentiation and proliferation. This protein has three isoforms (LAP1, LAP2, and LIP with different transcription activation potential. The role of female sex hormones in the expression pattern of C/EBPβ isoforms in the rat brain has not yet been described. In this study we demonstrate by western blot that the expression of the three C/EBPβ isoforms changes in different brain areas during the estrous cycle. In the cerebellum, LAP2 content diminished on diestrus and proestrus and LIP content diminished on proestrus and estrus days. In the prefrontal cortex, LIP content was higher on proestrus and estrus days. In the hippocampus, LAP isoforms presented a switch on diestrus day, since LAP1 content was the highest while that of LAP2 was the lowest. The LAP2 isoform was the most abundant one in all the three brain areas. The LAP/LIP ratio changed throughout the cycle and was tissue specific. These results suggest that C/EBPβ isoforms expression changes in a tissue-specific manner in the rat brain due to the changes in sex steroid hormone levels presented during the estrous cycle.

Characterization of ß-Galactosidase Isoforms from Bacillus circulans and Their Contribution to GOS Production

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Warmerdam, A.; Paudel, E.; Wanqing, J.; Boom, R.M.; Janssen, A.E.M.

2013-01-01

A ß-galactosidase preparation from Bacillus circulans consists of four isoforms called ß-gal-A, ß-gal-B, ß-gal-C, and ß-gal-D. These isoforms differ in lactose hydrolysis and galacto-oligosaccharide (GOS) synthesis at low substrate concentrations. For this reason, using a selection of the isoforms

Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms.

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Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M

2016-01-01

Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies' potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease.

Acidosis-mediated regulation of the NHE1 isoform of the Na⁺/H⁺ exchanger in renal cells.

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Odunewu, Ayodeji; Fliegel, Larry

2013-08-01

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that regulates intracellular pH by removing a proton in exchange for extracellular sodium. Renal tissues are subject to metabolic and respiratory acidosis, and acidosis has been shown to acutely activate NHE1 activity in other cell types. We examined if NHE1 is activated by acute acidosis in HEK293 and Madin-Darby canine kidney (MDCK) cells. Acute sustained intracellular acidosis (SIA) activated NHE1 in both cell types. We expressed wild-type and mutant NHE1 cDNAs in MDCK cells. All the cDNAs had a L163F/G174S mutation, which conferred a 100-fold resistance to EMD87580, an NHE1-specific inhibitor. We assayed exogenous NHE1 activity while inhibiting endogenous activity with EMD87580 and while inhibiting the NHE3 isoform of the Na⁺/H⁺ exchanger using the isoform-specific inhibitor S3226. We examined the activation and phosphorylation of the wild-type and mutant NHE1 proteins in response to SIA. In MDCK cells we demonstrated that the amino acids Ser⁷⁷¹, Ser⁷⁷⁶, Thr⁷⁷⁹, and Ser⁷⁸⁵ are important for NHE1 phosphorylation and activation after acute SIA. SIA activated ERK-dependent pathways in MDCK cells, and this was blocked by treatment with the MEK inhibitor U0126. Treatment with U0126 also blocked activation of NHE1 by SIA. These results suggest that acute acidosis activates NHE1 in mammalian kidney cells and that in MDCK cells this activation occurs through an ERK-dependent pathway affecting phosphorylation of a distinct set of amino acids in the cytosolic regulatory tail of NHE1.

Monolayer arrangement of fatty hydroxystearic acids on graphite: Influence of hydroxyl groups

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Medina, S. [Laboratorio de Rayos-X, Centro de Investigación Tecnología e Innovación, de la Universidad de Sevilla (CITIUS), Universidad de Sevilla, Avenida Reina Mercedes, 4B. 41012, Sevilla (Spain); Benítez, J.J.; Castro, M.A. [Instituto de Ciencia de Materiales de Sevilla, Consejo Superior de Investigaciones Científicas-Universidad de Sevilla, Avenida Américo Vespucio, 49. 41092, Sevilla (Spain); Cerrillos, C. [Servicio de Microscopía, Centro de Investigación Tecnología e Innovación, de la Universidad de Sevilla (CITIUS), Universidad de Sevilla, Avenida Reina Mercedes, 4B. 41012, Sevilla (Spain); Millán, C. [Instituto de Ciencia de Materiales de Sevilla, Consejo Superior de Investigaciones Científicas-Universidad de Sevilla, Avenida Américo Vespucio, 49. 41092, Sevilla (Spain); Alba, M.D., E-mail: alba@icmse.csic.es [Instituto de Ciencia de Materiales de Sevilla, Consejo Superior de Investigaciones Científicas-Universidad de Sevilla, Avenida Américo Vespucio, 49. 41092, Sevilla (Spain)

2013-07-31

Previous studies have indicated that long-chain linear carboxylic acids form commensurate packed crystalline monolayers on graphite even at temperatures above their melting point. This study examines the effect on the monolayer formation and structure of adding one or more secondary hydroxyl, functional groups to the stearic acid skeleton (namely, 12-hydroxystearic and 9,10-dihydroxystearic acid). Moreover, a comparative study of the monolayer formation on recompressed and monocrystalline graphite has been performed through X-ray diffraction (XRD) and Scanning Tunneling Microscopy (STM), respectively. The Differential Scanning Calorimetry (DSC) and XRD data were used to confirm the formation of solid monolayers and XRD data have provided a detailed structural analysis of the monolayers in good correspondence with obtained STM images. DSC and XRD have demonstrated that, in stearic acid and 12-hydroxystearic acid adsorbed onto graphite, the monolayer melted at a higher temperature than the bulk form of the carboxylic acid. However, no difference was observed between the melting point of the monolayer and the bulk form for 9,10-dihydroxystearic acid adsorbed onto graphite. STM results indicated that all acids on the surface have a rectangular p2 monolayer structure, whose lattice parameters were uniaxially commensurate on the a-axis. This structure does not correlate with the initial structure of the pure compounds after dissolving, but it is conditioned to favor a) hydrogen bond formation between the carboxylic groups and b) formation of hydrogen bonds between secondary hydroxyl groups, if spatially permissible. Therefore, the presence of hydroxyl functional groups affects the secondary structure and behavior of stearic acid in the monolayer. - Highlights: • Hydroxyl functional groups affect structure and behavior of acids in the monolayer. • Acids on the surface have a rectangular p2 monolayer structure. • Lattice parameters of acids are uniaxially

Analysis of heparanase isoforms and cathepsin B in the plasma of patients with gastrointestinal carcinomas: analytical cross-sectional study

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Carina Mucciolo Melo

Full Text Available CONTEXT AND OBJECTIVE: Heparanase-1 degrades heparan sulfate and has been correlated with tumor progression. Although the isoform heparanase-2 has no catalytic activity, it seems to be important for modulating heparanase-1 activity. Cathepsin B is a proteinase involved in tumor metastasis. The aim of this study was to analyze heparanase isoform expression and cathepsin B activity in plasma samples from patients with gastrointestinal carcinomas, compared with healthy individuals (control group. DESIGN AND SETTING: This was an analytical cross-sectional study. Peripheral blood samples were collected at a Brazilian public hospital, from 21 patients with histopathological diagnoses of gastrointestinal carcinomas and from 43 healthy individuals. The analyses were performed in two Brazilian medical schools. METHODS: Heparanase isoforms were identified and quantified in plasma samples by means of Western blot. The enzymatic activities of heparanase-1 and cathepsin B were also measured. RESULTS: The results demonstrated that the expression of both heparanase isoforms was significantly greater in plasma samples from gastrointestinal carcinoma patients, compared with the control group. Logistic regression analysis showed that increased heparanase-1 and heparanase-2 expression was exclusively dependent on the tumor. There was a significant increase in heparanase-1 and cathepsin B activity in the patients' plasma. CONCLUSION: Overexpression of heparanase-1 and heparanase-2, along with increased heparanase-1 and cathepsin B activity in plasma, is associated with the diagnosis of gastrointestinal carcinoma. These findings provide support for using non-invasive assays (plasma samples as an auxiliary method for diagnosing gastrointestinal tumors.

Functions of PDE3 Isoforms in Cardiac Muscle

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Movsesian, Matthew; Ahmad, Faiyaz

2018-01-01

Isoforms in the PDE3 family of cyclic nucleotide phosphodiesterases have important roles in cyclic nucleotide-mediated signalling in cardiac myocytes. These enzymes are targeted by inhibitors used to increase contractility in patients with heart failure, with a combination of beneficial and adverse effects on clinical outcomes. This review covers relevant aspects of the molecular biology of the isoforms that have been identified in cardiac myocytes; the roles of these enzymes in modulating cAMP-mediated signalling and the processes mediated thereby; and the potential for targeting these enzymes to improve the profile of clinical responses. PMID:29415428

Each individual isoform of the dopamine D2 receptor protects from lactotroph hyperplasia.

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Radl, Daniela; De Mei, Claudia; Chen, Eric; Lee, Hyuna; Borrelli, Emiliana

2013-06-01

Dopamine acting through D2 receptors (D2Rs) controls lactotroph proliferation and prolactin (PRL) levels. Ablation of this receptor in mice results in lactotroph hyperplasia and prolactinomas in aged females. Alternative splicing of the Drd2 gene generates 2 independent isoforms, a long (D2L) and a short (D2S) isoform, which are present in all D2R-expressing cells. Here, we addressed the role of D2L and D2S on lactotroph physiology through the generation and analysis of D2S-null mice and their comparison with D2L-null animals. These mice represent a valuable tool with which to investigate dopamine-dependent isoform-specific signaling in the pituitary gland. We sought to assess the existence of a more prominent role of D2L or D2S in controlling PRL expression and lactotroph hyperplasia. Importantly, we found that D2L and D2S are specifically linked to independent transduction pathways in the pituitary. D2L-mediated signaling inhibits the AKT/protein kinase B kinase activity whereas D2S, in contrast, is required for the activation of the ERK 1/2 pathway. Under normal conditions, presence of only 1 of the 2 D2R isoforms in vivo prevents hyperprolactinemia, formation of lactotroph's hyperplasia, and tumorigenesis that is observed when both isoforms are deleted as in D2R-/- mice. However, the protective function of the single D2R isoforms is overridden when single isoform-knockout mice are challenged by chronic estrogen treatments as they show increased PRL production and lactotroph hyperplasia. Our study indicates that signaling from each of the D2R isoforms is sufficient to maintain lactotroph homeostasis in physiologic conditions; however, signaling from both is necessary in conditions simulating pathologic states.

Robust stratification of breast cancer subtypes using differential patterns of transcript isoform expression.

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Thomas P Stricker

2017-03-01

Full Text Available Breast cancer, the second leading cause of cancer death of women worldwide, is a heterogenous disease with multiple different subtypes. These subtypes carry important implications for prognosis and therapy. Interestingly, it is known that these different subtypes not only have different biological behaviors, but also have distinct gene expression profiles. However, it has not been rigorously explored whether particular transcriptional isoforms are also differentially expressed among breast cancer subtypes, or whether transcript isoforms from the same sets of genes can be used to differentiate subtypes. To address these questions, we analyzed the patterns of transcript isoform expression using a small set of RNA-sequencing data for eleven Estrogen Receptor positive (ER+ subtype and fourteen triple negative (TN subtype tumors. We identified specific sets of isoforms that distinguish these tumor subtypes with higher fidelity than standard mRNA expression profiles. We found that alternate promoter usage, alternative splicing, and alternate 3'UTR usage are differentially regulated in breast cancer subtypes. Profiling of isoform expression in a second, independent cohort of 68 tumors confirmed that expression of splice isoforms differentiates breast cancer subtypes. Furthermore, analysis of RNAseq data from 594 cases from the TCGA cohort confirmed the ability of isoform usage to distinguish breast cancer subtypes. Also using our expression data, we identified several RNA processing factors that were differentially expressed between tumor subtypes and/or regulated by estrogen receptor, including YBX1, YBX2, MAGOH, MAGOHB, and PCBP2. RNAi knock-down of these RNA processing factors in MCF7 cells altered isoform expression. These results indicate that global dysregulation of splicing in breast cancer occurs in a subtype-specific and reproducible manner and is driven by specific differentially expressed RNA processing factors.

Effects of Andrographis paniculata and Orthosiphon stamineus Extracts on the Glucuronidation of 4-Methylumbelliferone in Human UGT Isoforms

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Sabariah Ismail

2010-05-01

Full Text Available The effects of Andrographis paniculata and Orthosiphon stamineus extracts on the in vitro glucuronidation of 4-methylumbelliferone (4MU by recombinant human UGTs, UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7 and UGT2B15 were determined. The potential inhibitory effects of both of the extracts on the activity of each of the UGT isoforms were investigated using 4MU as the substrate. Incubations contained UDP-glucuronic acid (UDPGA as the cofactor, MgCl2, cell lysate of respective isoform, and 4MU at the approximate apparent Km or S50 value of each isoform. Final concentrations of Andrographis paniculata and Orthosiphon stamineus extracts used were 0.025, 0.25, 2.5, 25 and 50 μg/mL and 0.01, 0.10, 1.0, 10 and 50 μg/mL respectively. Both extracts variably inhibited the activity of most of the isoforms in a concentration dependent manner. Andrographis paniculata extract was the better inhibitor of all the isoforms studied (IC50 1.70 μg/mL for UGT1A3, 2.57 μg/mL for UGT1A8, 2.82 μg/mL for UGT2B7, 5.00 μg/mL for UGT1A1, 5.66 μg/mL for UGT1A6, 9.88 μg/mL for UGT1A7 and 15.66 μg/mL for UGT1A10. Both extracts showed less than 70% inhibition of UGT2B15, so the IC50 values were >50μg/mL. The inhibition of human UGTs by Andrographis paniculata and Orthosiphon stamineus extracts in vitro suggests a potential for drug-herbal extract interactions in the therapeutic setting.

Estimation of Physical Properties of Amino Acids by Group-Contribution Method

DEFF Research Database (Denmark)

Jhamb, Spardha Virendra; Liang, Xiaodong; Gani, Rafiqul

2018-01-01

In this paper, we present group-contribution (GC) based property models for estimation of physical properties of amino acids using their molecular structural information. The physical properties modelled in this work are normal melting point (Tm), aqueous solubility (Ws), and octanol....../water partition coefficient (Kow) of amino acids. The developed GC-models are based on the published GC-method by Marrero and Gani (J. Marrero, R. Gani, Fluid Phase Equilib. 2001, 183-184, 183-208) with inclusion of new structural parameters (groups and molecular weight of compounds). The main objective...... of introducing these new structural parameters in the GC-model is to provide additional structural information for amino acids having large and complex structures and thereby improve predictions of physical properties of amino acids. The group-contribution values were calculated by regression analysis using...

Trimesic acid dimethyl sulfoxide solvate: space group revision

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Sylvain Bernès

2008-07-01

Full Text Available The structure of the title solvate, C9H6O6·C2H6OS, was determined 30 years ago [Herbstein, Kapon & Wasserman (1978. Acta Cryst. B34, 1613–1617], with data collected at room temperature, and refined in the space group P21. The present redetermination, based on high-resolution diffraction data, shows that the actual space group is more likely to be P21/m. The crystal structure contains layers of trimesic acid molecules lying on mirror planes. A mirror plane also passes through the S and O atoms of the solvent molecule. The molecules in each layer are interconnected through strong O—H...O hydrogen bonds, forming a two-dimensional supramolecular network within each layer. The donor groups are the hydroxyls of the trimesic acid molecules, while the acceptors are the carbonyl or the sulfoxide O atoms.

Proliferation marker pKi-67 occurs in different isoforms with various cellular effects.

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Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Finniss, Susan; Bögler, Oliver; Duchrow, Michael

2004-04-15

The Ki-67 antigen, pKi-67, is a commonly used proliferation marker in research and pathology. It has been recognized that the protein exists in two different splice variants that differ in one exon. In the current work, we present three new splice variants of human pKi-67 consisting of two naturally occurring isoforms and one atypical version. Additionally, data is presented indicating that alternative splicing of the pKi-67 N-terminus is common in tumor cell lines. Analyzing 93 tissues mainly consisting of brain tumor specimens, we found evidence that long and short isoform can be expressed independently of each other. Induction of mitosis in human peripheral blood mononuclear cells revealed that short pKi-67 appears earlier in the cell cycle than the long isoform and reaches its expression maximum when transcription of the latter sets in. Finally, transfection of mammalian culture cells with exon 7 (specific for the long pKi-67 isoform and not present in the short isoform) in a tetracycline regulated expression system decreased the rate of cell proliferation without affecting the cell cycle. In summary, we present evidence that the pKi-67 N-terminus is differentially spliced resulting in at least five different isoforms with different functions. Copyright 2004 Wiley-Liss, Inc.

Improved mass spectrometry assay for plasma hepcidin: detection and characterization of a novel hepcidin isoform.

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Coby M M Laarakkers

Full Text Available Mass spectrometry (MS-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25(+40 isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF platforms. As expected, implementation of hepcidin-25(+40 as internal standard in our weak cation exchange (WCX TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9 with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and

Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

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Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, Volker; Rülicke, Thomas; Rathkolb, Birgit; Hans, Wolfgang; Bohla, Alexander; Eickelberg, Oliver; Stoeger, Tobias; Wolf, Eckhard; Yildirim, Ali Önder; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; Hozák, Pavel

2013-01-01

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

Overexpression of EMMPRIN Isoform 2 Is Associated with Head and Neck Cancer Metastasis

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Huang, Zhiquan; Tan, Ning; Guo, Weijie; Wang, Lili; Li, Haigang; Zhang, Tianyu; Liu, Xiaojia; Xu, Qin; Li, Jinsong; Guo, Zhongmin

2014-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN), a plasma membrane protein of the immunoglobulin (Ig) superfamily, has been reported to promote cancer cell invasion and metastasis in several human malignancies. However, the roles of the different EMMPRIN isoforms and their associated mechanisms in head and neck cancer progression remain unknown. Using quantitative real-time PCR, we found that EMMPRIN isoform 2 (EMMPRIN-2) was the only isoform that was overexpressed in both head and n...

Isoform-selective regulation of glycogen phosphorylase by energy deprivation and phosphorylation in astrocytes.

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Müller, Margit S; Pedersen, Sofie E; Walls, Anne B; Waagepetersen, Helle S; Bak, Lasse K

2015-01-01

Glycogen phosphorylase (GP) is activated to degrade glycogen in response to different stimuli, to support both the astrocyte's own metabolic demand and the metabolic needs of neurons. The regulatory mechanism allowing such a glycogenolytic response to distinct triggers remains incompletely understood. In the present study, we used siRNA-mediated differential knockdown of the two isoforms of GP expressed in astrocytes, muscle isoform (GPMM), and brain isoform (GPBB), to analyze isoform-specific regulatory characteristics in a cellular setting. Subsequently, we tested the response of each isoform to phosphorylation, triggered by incubation with norepinephrine (NE), and to AMP, increased by glucose deprivation in cells in which expression of one GP isoform had been silenced. Successful knockdown was demonstrated on the protein level by Western blot, and on a functional level by determination of glycogen content showing an increase in glycogen levels following knockdown of either GPMM or GPBB. NE triggered glycogenolysis within 15 min in control cells and after GPBB knockdown. However, astrocytes in which expression of GPMM had been silenced showed a delay in response to NE, with glycogen levels significantly reduced only after 60 min. In contrast, allosteric activation of GP by AMP, induced by glucose deprivation, seemed to mainly affect GPBB, as only knockdown of GPBB, but not of GPMM, delayed the glycogenolytic response to glucose deprivation. Our results indicate that the two GP isoforms expressed in astrocytes respond to different physiological triggers, therefore conferring distinct metabolic functions of brain glycogen. © 2014 Wiley Periodicals, Inc.

Quantitative mass spectrometry reveals changes in SNAP-25 isoforms in schizophrenia

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Barakauskas, Vilte E; Moradian, Annie; Barr, Alasdair M.; Beasley, Clare L; Rosoklija, Gorazd; Mann, J John; Ilievski, Boro; Stankov, Aleksandar; Dwork, Andrew J; Falkai, Peter; Morin, Gregg B; Honer, William G

2016-01-01

SNAP-25 and syntaxin are presynaptic terminal SNARE proteins altered in amount and function in schizophrenia. In the ventral caudate, we observed 32% lower SNAP-25 and 26% lower syntaxin, but greater interaction between the two proteins using an in vitro assay. SNAP-25 has two isoforms, SNAP-25A and B, differing by only 9 amino acids, but with different effects on neurotransmission. A quantitative mass spectrometry assay was developed to measure total SNAP-25, and proportions of SNAP-25A and B. The assay had a good linear range (50- to 150-fold) and coefficient of variation (4.5%). We studied ventral caudate samples from patients with schizophrenia (n=15) previously reported to have lower total SNAP-25 than controls (n=13). We confirmed 27% lower total SNAP-25 in schizophrenia, and observed 31% lower SNAP-25A (P = 0.002) with 20% lower SNAP-25B amounts (P = 0.10). Lower SNAP-25A amount correlated with greater SNAP-25-syntaxin protein-protein interactions (r = -0.41, P = 0.03); the level of SNAP-25B did not. Administration of haloperidol or clozapine to rats did not mimic the changes found in schizophrenia. The findings suggest that lower levels of SNAP-25 in schizophrenia may represent a greater effect of the illness on the SNAP-25A isoform. This in turn could contribute to the greater interaction between SNAP25 and syntaxin, and possibly disturb neurotransmission in the illness. PMID:26971072

The combined expression patterns of Ikaros isoforms characterize different hematological tumor subtypes.

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Carlos A Orozco

Full Text Available A variety of genetic alterations are considered hallmarks of cancer development and progression. The Ikaros gene family, encoding for key transcription factors in hematopoietic development, provides several examples as genetic defects in these genes are associated with the development of different types of leukemia. However, the complex patterns of expression of isoforms in Ikaros family genes has prevented their use as clinical markers. In this study, we propose the use of the expression profiles of the Ikaros isoforms to classify various hematological tumor diseases. We have standardized a quantitative PCR protocol to estimate the expression levels of the Ikaros gene exons. Our analysis reveals that these levels are associated with specific types of leukemia and we have found differences in the levels of expression relative to five interexonic Ikaros regions for all diseases studied. In conclusion, our method has allowed us to precisely discriminate between B-ALL, CLL and MM cases. Differences between the groups of lymphoid and myeloid pathologies were also identified in the same way.

Interplay between PTB and miR-1285 at the p53 3'UTR modulates the levels of p53 and its isoform Δ40p53α.

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Katoch, Aanchal; George, Biju; Iyyappan, Amrutha; Khan, Debjit; Das, Saumitra

2017-09-29

p53 and its translational isoform Δ40p53 are involved in many important cellular functions like cell cycle, cell proliferation, differentiation and metabolism. Expression of both the isoforms can be regulated at different steps. In this study, we explored the role of 3'UTR in regulating the expression of these two translational isoforms. We report that the trans acting factor, Polypyrimidine Tract Binding protein (PTB), also interacts specifically with 3'UTR of p53 mRNA and positively regulates expression of p53 isoforms. Our results suggest that there is interplay between miRNAs and PTB at the 3'UTR under normal and stress conditions like DNA damage. Interestingly, PTB showed some overlapping binding regions in the p53 3'UTR with miR-1285. In fact, knockdown of miR-1285 as well as expression of p53 3'UTR with mutated miR-1285 binding sites resulted in enhanced association of PTB with the 3'UTR, which provides mechanistic insights of this interplay. Taken together, the results provide a plausible molecular basis of how the interplay between miRNAs and the PTB protein at the 3'UTR can play pivotal role in fine tuning the expression of the two p53 isoforms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Kalrn promoter usage and isoform expression respond to chronic cocaine exposure

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Ma Xin-Ming

2011-02-01

Full Text Available Abstract Background The long-term effects of cocaine on behavior are accompanied by structural changes in excitatory glutamatergic synapses onto the medium spiny neurons of the striatum. The Kalrn gene encodes several functionally distinct isoforms; these multidomain guanine nucleotide exchange factors (GEFs contain additional domains known to interact with phosphatidylinositides as well as with a number of different proteins. Through their activation of Rho proteins and their interactions with other proteins, the different Kalirin isoforms affect cytoskeletal organization. Chronic exposure of adult male rodents to cocaine increases levels of Kalirin 7 in the striatum. When exposed chronically to cocaine, mice lacking Kalirin 7, the major adult isoform, fail to show an increase in dendritic spine density in the nucleus accumbens, show diminished place preference for cocaine, and exhibit increased locomotor activity in response to cocaine. Results The use of alternate promoters and 3'-terminal exons of the mouse Kalrn gene were investigated using real-time quantitative polymerase chain reaction. While the two most distal full-length Kalrn promoters are used equally in the prefrontal cortex, the more proximal of these promoters accounts for most of the transcripts expressed in the nucleus accumbens. The 3'-terminal exon unique to the Kalirin 7 isoform accounts for a greater percentage of the Kalrn transcripts in prefrontal cortex than in nucleus accumbens. Western blot analyses confirmed these differences. Chronic cocaine treatment increases usage of the promoter encoding the Δ-Kalirin isoforms but does not alter full-length Kalirin promoter usage. Usage of the 3'-terminal exon unique to Kalirin 7 increases following chronic cocaine exposure. Conclusions Kalrn promoter and 3'-terminal exon utilization are region-specific. In the nucleus accumbens, cocaine-mediated alterations in promoter usage and 3'-terminal exon usage favor expression of

Effect of adult onset hypothyroidism on behavioral parameters and acetylcholinesterase isoforms activity in specific brain regions of male mice.

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Vasilopoulou, Catherine G; Constantinou, Caterina; Giannakopoulou, Dimitra; Giompres, Panagiotis; Margarity, Marigoula

2016-10-01

Thyroid hormones (TH) are essential for normal development and function of mammalian central nervous system (CNS); TH dysregulation has been implicated in several cognitive and behavioral deficits related to dysfunctions of neurotransmitter systems. In the present study, we investigated the effects of adult onset hypothyroidism on the activity of acetylcholinesterase (AChE) and on related behavioral parameters. For this purpose we used adult male Balb/cJ mice that were divided randomly into euthyroid and hypothyroid animal groups. Animals were rendered hypothyroid through administration of 1% w/v KClO4 in their drinking water for 8weeks. At the end of the treatment, learning/memory procedures were examined through step-through passive avoidance task while fear/anxiety was assessed using elevated plus-maze (EPM) and open-field (OF) tests. AChE activity was determined colorimetrically in two different fractions, salt-soluble fraction (SS) (containing mainly the G1 isoform) and detergent-soluble fraction (DS) (containing mainly the G4 isoform) in cerebral cortex, cerebellum, midbrain, hippocampus and striatum. Our results indicate that adult onset hypothyroidism caused significant memory impairment and increased fear/anxiety. Moreover, the activity of both isoforms of AChE was reduced in all brain regions examined in a brain region- and isoform-specific manner. Copyright © 2016. Published by Elsevier Inc.

Hydrogen-bond acidic functionalized carbon nanotubes (CNTs) with covalently-bound hexafluoroisopropanol groups

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Fifield, Leonard S.; Grate, Jay W.

2010-06-01

Fluorinated hydrogen-bond acidic groups are directly attached to the backbone of single walled carbon nanotubes (SWCNTs) without the introduction of intermediate electron donating surface groups. Hexafluoroalcohol functional groups are exceptionally strong hydrogen bond acids, and are added to the nanotube surface using the aryl diazonium approach to create hydrogen-bond acidic carbon nanotube (CNT) surfaces. These groups can promote strong hydrogen-bonding interactions with matrix materials in composites or with molecular species to be concentrated and sensed. In the latter case, this newly developed material is expected to find useful application in chemical sensors and in CNT-based preconcentrator devices for the detection of pesticides, chemical warfare agents and explosives.

mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome

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Beatriz Puisac

2017-02-01

Full Text Available Cornelia de Lange syndrome (CdLS is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction. Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys, showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers.

Alternative NF-κB Isoforms in the Drosophila Neuromuscular Junction and Brain.

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Bo Zhou

Full Text Available The Drosophila NF-κB protein Dorsal is expressed at the larval neuromuscular junction, where its expression appears unrelated to known Dorsal functions in embryonic patterning and innate immunity. Using confocal microscopy with domain-specific antisera, we demonstrate that larval muscle expresses only the B isoform of Dorsal, which arises by intron retention. We find that Dorsal B interacts with and stabilizes Cactus at the neuromuscular junction, but exhibits Cactus independent localization and an absence of detectable nuclear translocation. We further find that the Dorsal-related immune factor Dif encodes a B isoform, reflecting a conservation of B domains across a range of insect NF-κB proteins. Carrying out mutagenesis of the Dif locus via a site-specific recombineering approach, we demonstrate that Dif B is the major, if not sole, Dif isoform in the mushroom bodies of the larval brain. The Dorsal and Dif B isoforms thus share a specific association with nervous system tissues as well as an alternative protein structure.

Redundancy of IL-1 Isoform Signaling and Its Implications for Arterial Remodeling.

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Marina Beltrami-Moreira

Full Text Available Mice deficient in IL-1 receptor 1 (hence unresponsive to both IL-1 isoforms α and β have impaired expansive arterial remodeling due to diminished expression of matrix-degrading enzymes, especially MMP-3. Emergence of IL-1 as a target in cardiovascular disease prompted the investigation of the redundancy of IL-1α and IL-1β in the induction of MMP-3 and other matrix-remodeling enzymes in human cells.Human primary vascular smooth muscle cells (VSMCs and carotid endarterectomy specimens were stimulated with equimolar concentrations of IL-1α or IL-1β and analyzed protease expression by immunoblot and ELISA. Either IL-1α or IL-1β increased the expression of pro-MMP-3 in VSMCs, facilitated VSMC migration through Matrigel, and induced MMP-3 production in specimens from atheromatous plaques. VSMCs also secreted MMP-1 and Cathepsin S (CatS upon stimulation with IL-1α or IL-1β. IL-1 isoforms similarly increased MMP-1 and MMP-9 expression in carotid endarterectomy specimens. We examined the expression of MMP-3 and IL-1 isoforms by immunostaining of carotid atheromata, calculated the % positive areas, and tested associations by linear regression. MMP-3 colocalized with IL-1 isoforms in atheromata. MMP-3+ area in plaques positively associated with IL-1α+ (R2 = 0.61, P<0.001 and with IL-1β + areas (R2 = 0.68, P<0.001. MMP-3+ area within atheroma also associated with CD68+ area, but not with α-smooth muscle actin area.Either IL-1α or IL-1β can induce the expression of enzymes implicated in remodeling of the arterial extracellular matrix, and facilitate human VSMC migration in vitro. Human atheromata contain both IL-1 isoforms in association with immunoreactive MMP-3. This redundancy of IL-1 isoforms suggests that selective blocking of one IL-1 isoform should not impair expansive arterial remodeling, a finding with important clinical implications for therapeutic targeting of IL-1 in atherosclerosis.

Novel gamma-carboxyglutamic acid-containing peptides from the venom of Conus textile

DEFF Research Database (Denmark)

Czerwiec, Eva; Kalume, Dario E; Roepstorff, Peter

2006-01-01

-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding...

The ROCK isoforms differentially regulate the morphological characteristics of carcinoma cells.

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Jerrell, Rachel J; Leih, Mitchell J; Parekh, Aron

2017-06-26

Rho-associated kinase (ROCK) activity drives cell migration via actomyosin contractility. During invasion, individual cancer cells can transition between 2 modes of migration, mesenchymal and amoeboid. Changes in ROCK activity can cause a switch between these migration phenotypes which are defined by distinct morphologies. However, recent studies have shown that the ROCK isoforms are not functionally redundant as previously thought. Therefore, it is unclear whether the ROCK isoforms play different roles in regulating migration phenotypes. Here, we found that ROCK1 and ROCK2 differentially regulate carcinoma cell morphology resulting in intermediate phenotypes that share some mesenchymal and amoeboid characteristics. These findings suggest that the ROCK isoforms play unique roles in the phenotypic plasticity of mesenchymal carcinoma cells which may have therapeutic implications.

Nubbin isoform antagonism governs Drosophila intestinal immune homeostasis.

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Bo G Lindberg

2018-03-01

Full Text Available Gut immunity is regulated by intricate and dynamic mechanisms to ensure homeostasis despite a constantly changing microbial environment. Several regulatory factors have been described to participate in feedback responses to prevent aberrant immune activity. Little is, however, known about how transcriptional programs are directly tuned to efficiently adapt host gut tissues to the current microbiome. Here we show that the POU/Oct gene nubbin (nub encodes two transcription factor isoforms, Nub-PB and Nub-PD, which antagonistically regulate immune gene expression in Drosophila. Global transcriptional profiling of adult flies overexpressing Nub-PB in immunocompetent tissues revealed that this form is a strong transcriptional activator of a large set of immune genes. Further genetic analyses showed that Nub-PB is sufficient to drive expression both independently and in conjunction with nuclear factor kappa B (NF-κB, JNK and JAK/STAT pathways. Similar overexpression of Nub-PD did, conversely, repress expression of the same targets. Strikingly, isoform co-overexpression normalized immune gene transcription, suggesting antagonistic activities. RNAi-mediated knockdown of individual nub transcripts in enterocytes confirmed antagonistic regulation by the two isoforms and that both are necessary for normal immune gene transcription in the midgut. Furthermore, enterocyte-specific Nub-PB expression levels had a strong impact on gut bacterial load as well as host lifespan. Overexpression of Nub-PB enhanced bacterial clearance of ingested Erwinia carotovora carotovora 15. Nevertheless, flies quickly succumbed to the infection, suggesting a deleterious immune response. In line with this, prolonged overexpression promoted a proinflammatory signature in the gut with induction of JNK and JAK/STAT pathways, increased apoptosis and stem cell proliferation. These findings highlight a novel regulatory mechanism of host-microbe interactions mediated by antagonistic

Enhanced expression of two discrete isoforms of matrix metalloproteinase-2 in experimental and human diabetic nephropathy.

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Sang Soo Kim

Full Text Available We recently reported on the enhanced expression of two isoforms of matrix metalloproteinase-2 (MMP-2 in human renal transplantation delayed graft function. These consist of the conventional secreted, full length MMP-2 isoform (FL-MMP-2 and a novel intracellular N-Terminal Truncated isoform (NTT-MMP-2 generated by oxidative stress-mediated activation of an alternate promoter in the MMP-2 first intron. Here we evaluated the effect of hyperglycemia and diabetes mellitus on the in vitro and in vivo expression of the two MMP-2 isoforms.We quantified the abundance of the FL-MMP-2 and NTT-MMP-2 transcripts by qPCR in HK2 cells cultured in high glucose or 4-hydroxy-2-hexenal (HHE and tested the effects of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC. The streptozotocin (STZ murine model of Type I diabetes mellitus and renal biopsies of human diabetic nephropathy were used in this study.Both isoforms of MMP-2 in HK2 cells were upregulated by culture in high glucose or with HHE. PDTC treatment did not suppress high glucose-mediated FL-MMP-2 expression but potently inhibited NTT-MMP-2 expression. With STZ-treated mice, renal cortical expression of both isoforms was increased (FL-MMP-2, 1.8-fold; NTT-MMP-2, greater than 7-fold. Isoform-specific immunohistochemical staining revealed low, but detectable levels of the FL-MMP-2 isoform in controls, while NTT-MMP-2 was not detected. While there was a modest increase in tubular epithelial cell staining for FL-MMP-2 in STZ-treated mice, NTT-MMP-2 was intensely expressed in a basolateral pattern. FL-MMP-2 and NTT-MMP-2 isoform expression as quantified by qPCR were both significantly elevated in renal biopsies of human diabetic nephropathy (12-fold and 3-fold, respectively.The expression of both isoforms of MMP-2 was enhanced in an experimental model of diabetic nephropathy and in human diabetic nephropathy. Selective MMP-2 isoform inhibition could offer a novel approach for the treatment of diabetic renal

Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G

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2011-01-01

Background Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA) comprises ~530 residues, the G isoform (MSG) is ~730 residues, and this third isoform (MSH-halophilic) is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea. Results We have solved the structure of a malate synthase H (MSH) isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a β8/α8 (TIM) barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A. Conclusions The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C-terminal domain of the H

Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development

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Kim, Kee K.; Adelstein, Robert S.; Kawamoto, Sachiyo, E-mail: kawamots@mail.nih.gov

2014-08-08

Highlights: • Protein stability of Rbfox3 splice isoforms is differentially regulated. • Rbfox3-d31, an Rbfox3 isoform lacking the RRM, is highly susceptible to degradation. • The protein stability of Rbfox3-d31 is regulated by the ubiquitin–proteasome pathway. • Rbfox3-d31 inhibits the nuclear localization of Rbfox2. • Rbfox3-d31 inhibits the splicing activity of Rbfox2. - Abstract: Rbfox3, a neuron-specific RNA-binding protein, plays an important role in neuronal differentiation during development. An isoform Rbfox3-d31, which excludes the 93-nucleotide cassette exon within the RNA recognition motif of chicken Rbfox3, has been previously identified. However, the cellular functions of Rbfox3-d31 remain largely unknown. Here we find that Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin–proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with full-length Rbfox3. Furthermore, suppression of Rbfox3-d31 protein degradation with the proteasome inhibitor MG132 attenuates the splicing activity of another Rbfox family member Rbfox2 by altering the subcellular localization of Rbfox2. These results suggest that Rbfox3-d31 functions as a repressor for the splicing activity of the Rbfox family and its protein level is regulated in an isoform-specific manner in vivo.

Reduced peripheral expression of the glucocorticoid receptor α isoform in individuals with posttraumatic stress disorder: a cumulative effect of trauma burden.

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Gola, Hannah; Engler, Andrea; Morath, Julia; Adenauer, Hannah; Elbert, Thomas; Kolassa, Iris-Tatjana; Engler, Harald

2014-01-01

Posttraumatic stress disorder (PTSD) is a serious psychiatric condition that was found to be associated with altered functioning of the hypothalamic-pituitary-adrenal (HPA) axis and changes in glucocorticoid (GC) responsiveness. The physiological actions of GCs are primarily mediated through GC receptors (GR) of which isoforms with different biological activities exist. This study aimed to investigate whether trauma-experience and/or PTSD are associated with altered expression of GR splice variants. GRα and GRβ mRNA expression levels were determined by real-time quantitative PCR in whole blood samples of individuals with chronic and severe forms of PTSD (n = 42) as well as in ethnically matched reference subjects (non-PTSD, n = 35). Individuals suffering from PTSD exhibited significantly lower expression of the predominant and functionally active GRα isoform compared to non-PTSD subjects. This effect remained significant when accounting for gender, smoking, psychotropic medication or comorbid depression. Moreover, the GRα expression level was significantly negatively correlated with the number of traumatic event types experienced, both in the whole sample and within the PTSD patient group. Expression of the less abundant and non-ligand binding GRβ isoform was comparable between patient and reference groups. Reduced expression of the functionally active GRα isoform in peripheral blood cells of individuals with PTSD seems to be a cumulative effect of trauma burden rather than a specific feature of PTSD since non-PTSD subjects with high trauma load showed an intermediate phenotype between PTSD patients and individuals with no or few traumatic experiences.

Reduced peripheral expression of the glucocorticoid receptor α isoform in individuals with posttraumatic stress disorder: a cumulative effect of trauma burden.

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Hannah Gola

Full Text Available BACKGROUND: Posttraumatic stress disorder (PTSD is a serious psychiatric condition that was found to be associated with altered functioning of the hypothalamic-pituitary-adrenal (HPA axis and changes in glucocorticoid (GC responsiveness. The physiological actions of GCs are primarily mediated through GC receptors (GR of which isoforms with different biological activities exist. This study aimed to investigate whether trauma-experience and/or PTSD are associated with altered expression of GR splice variants. METHODS: GRα and GRβ mRNA expression levels were determined by real-time quantitative PCR in whole blood samples of individuals with chronic and severe forms of PTSD (n = 42 as well as in ethnically matched reference subjects (non-PTSD, n = 35. RESULTS: Individuals suffering from PTSD exhibited significantly lower expression of the predominant and functionally active GRα isoform compared to non-PTSD subjects. This effect remained significant when accounting for gender, smoking, psychotropic medication or comorbid depression. Moreover, the GRα expression level was significantly negatively correlated with the number of traumatic event types experienced, both in the whole sample and within the PTSD patient group. Expression of the less abundant and non-ligand binding GRβ isoform was comparable between patient and reference groups. CONCLUSIONS: Reduced expression of the functionally active GRα isoform in peripheral blood cells of individuals with PTSD seems to be a cumulative effect of trauma burden rather than a specific feature of PTSD since non-PTSD subjects with high trauma load showed an intermediate phenotype between PTSD patients and individuals with no or few traumatic experiences.

Troponin T isoform expression is modulated during Atlantic Halibut metamorphosis

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Llewellyn Lynda

2007-06-01

Full Text Available Abstract Background Flatfish metamorphosis is a thyroid hormone (TH driven process which leads to a dramatic change from a symmetrical larva to an asymmetrical juvenile. The effect of THs on muscle and in particular muscle sarcomer protein genes is largely unexplored in fish. The change in Troponin T (TnT, a pivotal protein in the assembly of skeletal muscles sarcomeres and a modulator of calcium driven muscle contraction, during flatfish metamophosis is studied. Results In the present study five cDNAs for halibut TnT genes were cloned; three were splice variants arising from a single fast TnT (fTnT gene; a fourth encoded a novel teleost specific fTnT-like cDNA (AfTnT expressed exclusively in slow muscle and the fifth encoded the teleost specific sTnT2. THs modified the expression of halibut fTnT isoforms which changed from predominantly basic to acidic isoforms during natural and T4 induced metamorphosis. In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment. Prior to and after metamorphosis no change in the dorso-ventral symmetry or temporal-spatial expression pattern of TnT genes and muscle fibre organization occurred in halibut musculature. Conclusion Muscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations. We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.

Expression of 14-3-3 protein isoforms in mouse oocytes, eggs and ovarian follicular development

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De Santanu

2012-01-01

Full Text Available Abstract Background The 14-3-3 (YWHA proteins are a highly conserved, ubiquitously expressed family of proteins. Seven mammalian isoforms of 14-3-3 are known (β, γ, ε, ζ, η, τ and, σ. These proteins associate with many intracellular proteins involved in a variety of cellular processes including regulation of the cell cycle, metabolism and protein trafficking. We are particularly interested in the role of 14-3-3 in meiosis in mammalian eggs and the role 14-3-3 proteins may play in ovarian function. Therefore, we examined the expression of 14-3-3 proteins in mouse oocyte and egg extracts by Western blotting after polyacrylamide gel electrophoresis, viewed fixed cells by indirect immunofluorescence, and examined mouse ovarian cells by immunohistochemical staining to study the expression of the different 14-3-3 isoforms. Results We have determined that all of the mammalian 14-3-3 isoforms are expressed in mouse eggs and ovarian follicular cells including oocytes. Immunofluorescence confocal microscopy of isolated oocytes and eggs confirmed the presence of all of the isoforms with characteristic differences in some of their intracellular localizations. For example, some isoforms (β, ε, γ, and ζ are expressed more prominently in peripheral cytoplasm compared to the germinal vesicles in oocytes, but are uniformly dispersed within eggs. On the other hand, 14-3-3η is diffusely dispersed in the oocyte, but attains a uniform punctate distribution in the egg with marked accumulation in the region of the meiotic spindle apparatus. Immunohistochemical staining detected all isoforms within ovarian follicles, with some similarities as well as notable differences in relative amounts, localizations and patterns of expression in multiple cell types at various stages of follicular development. Conclusions We found that mouse oocytes, eggs and follicular cells within the ovary express all seven isoforms of the 14-3-3 protein. Examination of the

Myosin heavy-chain isoforms in the flight and leg muscles of hummingbirds and zebra finches.

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Velten, Brandy P; Welch, Kenneth C

2014-06-01

Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25-60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to understand how contractile and morphological properties of avian muscle may be reflected in MHC expression. Isoforms of the hummingbird and zebra finch flight and hindlimb muscles were electrophoretically separated and compared with those of other avian species representing different contractile properties and fiber types. The flight muscles of the study species operate at drastically different contraction rates and are composed of different histochemically defined fiber types, yet each exhibited the same, single MHC isoform corresponding to the chicken adult fast isoform. Thus, despite quantitative differences in the contractile demands of flight muscles across species, this isoform appears necessary for meeting the performance demands of avian powered flight. Variation in flight muscle contractile performance across species may be due to differences in the structural composition of this conserved isoform and/or variation within other mechanically linked proteins. The leg muscles were more varied in their MHC isoform composition across both muscles and species. The disparity in hindlimb MHC expression between hummingbirds and the other species highlights previously observed differences in fiber type composition and thrust production during take-off. Copyright © 2014 the American Physiological Society.

Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity

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Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly; Capka, Vladimir; Chen, Jinyun; Chenail, Gregg; Cho, YoungShin; Dooley, Julia; Farsidjani, Ali; Fortin, Pascal; Kohls, Darcy; Kulathila, Raviraj; Lin, Fallon; McKay, Daniel; Rodrigues, Lindsey; Sage, David; Touré, B. Barry; van der Plas, Simon; Wright, Kirk; Xu, Ming; Yin, Hong; Levell, Julian; Pagliarini, Raymond A. (Novartis)

2017-03-01

Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 “regulatory segment,” which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

Pomegranate ( Punica granatum L.) expresses several nsLTP isoforms characterized by different immunoglobulin E-binding properties.

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Bolla, Michela; Zenoni, Sara; Scheurer, Stephan; Vieths, Stefan; San Miguel Moncin, Maria Del Mar; Olivieri, Mario; Antico, Andrea; Ferrer, Marta; Berroa, Felicia; Enrique, Ernesto; Avesani, Linda; Marsano, Francesco; Zoccatelli, Gianni

2014-01-01

Pomegranate allergy is associated with sensitization to non-specific lipid transfer proteins (nsLTPs). Our aim was to identify and characterize the non-specific nsLTPs expressed in pomegranate at the molecular level and to study their allergenic properties in terms of immunoglobulin E (IgE)-binding and cross-reactivity with peach nsLTP (Pru p 3). A non-equilibrium two-dimensional (2-D) electrophoretic approach based on acid-urea PAGE and sodium dodecyl sulfate PAGE was set up to separate pomegranate nsLTPs. Their immunoreactivity was tested by immunoblotting carried out with anti-Pru p 3 polyclonal antibodies and sera from pomegranate-allergic patients. For final identification, pomegranate nsLTPs were purified by chromatography and subjected to trypsin digestion and mass spectrometry (MS) analysis. For this purpose, the sequences obtained by cDNA cloning of three pomegranate nsLTPs were integrated in the database that was subsequently searched for MS data interpretation. Four nsLTPs were identified by 2-D immunoblotting. The detected proteins showed different IgE-binding capacity and partial cross-reactivity with Pru p 3. cDNA cloning and MS analyses led to the identification of three nsLTP isoforms with 66-68% amino acid sequence identity named Pun g 1.0101, Pun g 1.0201 and Pun g 1.0301. By 2-D electrophoresis, we could separate different nsLTP isoforms possessing different IgE-binding properties, which might reflect peculiar allergenic potencies. The contribution of Pru p 3 to prime sensitization is not central as in other plant nsLTPs. © 2014 S. Karger AG, Basel.

Laminin isoforms: biological roles and effects on the intracellular distribution of nuclear proteins in intestinal epithelial cells

International Nuclear Information System (INIS)

Turck, Natacha; Gross, Isabelle; Gendry, Patrick; Stutzmann, Jeanne; Freund, Jean-Noel; Kedinger, Michele; Simon-Assmann, Patricia; Launay, Jean-Francois

2005-01-01

Laminins are structurally and functionally major components of the extracellular matrix. Four isoforms of laminins (laminin-1, -2, -5 and -10) are expressed in a specific pattern along the crypt-villus axis of the intestine. Previous works indicated that expression of these isoforms is developmentally regulated and that laminins could modulate the behaviour of intestinal cells, but the exact role of each isoform remained unclear. Here, we report the first systematic analysis of the cellular functions of the four isoforms using the human colon adenocarcinoma Caco2/TC7 cell line as a model. We compared the respective abilities of each isoform to modulate adhesion, proliferation and differentiation of intestinal epithelial cells. We found that the isoforms were functionally distinct, with laminin-10 being the most adhesive substratum, laminin-2, laminin-5 and laminin-10 enhancing cellular proliferation and at the opposite, laminin-1 stimulating intestinal cell differentiation. To begin to characterise the molecular events induced by the different isoforms, we examined by immunofluorescence the intracellular distribution of several nuclear proteins, recently highlighted by a nuclear proteomic approach. We observed clear nucleocytoplasmic redistribution of these proteins, which depended on the laminin isoform. These results provide evidence for a distinct functional role of laminins in intestinal cell functions characterised by specific localisation of nuclear proteins

Synthesis and biological evaluation of 2-heteroarylthioalkanoic acid analogues of clofibric acid as peroxisome proliferator-activated receptor alpha agonists.

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Giampietro, Letizia; Ammazzalorso, Alessandra; Giancristofaro, Antonella; Lannutti, Fabio; Bettoni, Giancarlo; De Filippis, Barbara; Fantacuzzi, Marialuigia; Maccallini, Cristina; Petruzzelli, Michele; Morgano, Annalisa; Moschetta, Antonio; Amoroso, Rosa

2009-10-22

A series of 2-heteroarylthioalkanoic acids were synthesized through systematic structural modifications of clofibric acid and evaluated for human peroxisome proliferator-activated receptor alpha (PPARalpha) transactivation activity, with the aim of obtaining new hypolipidemic compounds. Some thiophene and benzothiazole derivatives showing a good activation of the receptor alpha were screened for activity against the PPARgamma isoform. The gene induction of selected compounds was also investigated in the human hepatoma cell line.

Expression of Metallothionein and Vascular Endothelial Growth Factor Isoforms in Breast Cancer Cells.

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Wierzowiecka, Barbara; Gomulkiewicz, Agnieszka; Cwynar-Zajac, Lucja; Olbromski, Mateusz; Grzegrzolka, Jedrzej; Kobierzycki, Christopher; Podhorska-Okolow, Marzenna; Dziegiel, Piotr

2016-01-01

Metallothioneins (MTs) are low-molecular-weight and cysteine-rich proteins that bind heavy metal ions and oxygen-free radicals. MTs are commonly expressed in various tissues of mammals and are involved in regulation of cell proliferation and differentiation, and may be engaged in angiogenesis. Expression of MTs has been studied in many cancer types, especially breast cancer. The research results indicate that MTs may play important, although not yet fully known, roles in cancer angiogenesis. The aim of this study was to analyze the level of gene expression of selected MT isoforms induced with zinc ions in correlation with vascular endothelial growth factor (VEGF) isoforms in in vitro models of breast cancer. The studies were carried out in three breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231). An epithelial cell line derived from normal breast tissue (Me16c) was used as a control. The levels of expression of selected MT isoforms and selected genes involved in angiogenesis were studied with real-time PCR. Expression of different MT isoforms was induced by zinc ions to differing degrees in individual breast cancer cell lines. An increase in the expression of some MT isoforms was associated with a slight increase in the level of expression of VEGFA. The research results may indicate certain correlation between an increased expression of selected MT isoforms and a pro-angiogenic factor VEGF in specific types of breast cancer cells. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

P120-catenin isoforms 1A and 3A differently affect invasion and proliferation of lung cancer cells

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Liu Yang; Dong Qianze; Zhao Yue; Dong Xinjun; Miao Yuan; Dai Shundong; Yang Zhiqiang; Zhang Di; Wang Yan; Li Qingchang; Zhao Chen; Wang Enhua

2009-01-01

Different isoforms of p120-catenin (p120ctn), a member of the Armadillo gene family, are variably expressed in different tissues as a result of alternative splicing and the use of multiple translation initiation codons. When expressed in cancer cells, these isoforms may confer different properties with respect to cell adhesion and invasion. We have previously reported that the p120ctn isoforms 1 and 3 were the most highly expressed isoforms in normal lung tissues, and their expression level was reduced in lung tumor cells. To precisely define their biological roles, we transfected p120ctn isoforms 1A and 3A into the lung cancer cell lines A549 and NCI-H460. Enhanced expression of p120ctn isoform 1A not only upregulated E-cadherin and β-catenin, but also downregulated the Rac1 activity, and as a result, inhibited the ability of cells to invade. In contrast, overexpression of p120ctn isoform 3A led to the inactivation of Cdc42 and the activation of RhoA, and had a smaller influence on invasion. However, we found that isoform 3A had a greater ability than isoform 1A in both inhibiting the cell cycle and reducing tumor cell proliferation. The present study revealed that p120ctn isoforms 1A and 3A differently regulated the adhesive, proliferative, and invasive properties of lung cancer cells through distinct mechanisms

Delayed polarization of mononuclear phagocyte transcriptional program by type I interferon isoforms

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Wang Ena

2005-06-01

Full Text Available Abstract Background Interferon (IFN-α is considered a key modulator of immunopathological processes through a signature-specific activation of mononuclear phagocytes (MPs. This study utilized global transcript analysis to characterize the effects of the entire type I IFN family in comparison to a broad panel of other cytokines on MP previously exposed to Lipopolysaccharide (LPS stimulation in vitro. Results Immature peripheral blood CD14+ MPs were stimulated with LPS and 1 hour later with 42 separate soluble factors including cytokines, chemokines, interleukins, growth factors and IFNs. Gene expression profiling of MPs was analyzed 4 and 9 hours after cytokine stimulation. Four hours after stimulation, the transcriptional analysis of MPs revealed two main classes of cytokines: one associated with the alternative and the other with the classical pathway of MP activation without a clear polarization of type I IFNs effects. In contrast, after 9 hours of stimulation most type I IFN isoforms induced a characteristic and unique transcriptional pattern separate from other cytokines. These "signature" IFNs included; IFN-β, IFN-α2b/α2, IFN-αI, IFN-α2, IFN-αC, IFN-αJ1, IFN-αH2, and INF-α4B and induced the over-expression of 44 genes, all of which had known functional relationships with IFN such as myxovirus resistance (Mx-1, Mx-2, and interferon-induced hepatitis C-associated microtubular aggregation protein. A second group of type I IFNs segregated separately and in closer association with the type II IFN-γ. The phylogenetic relationship of amino acid sequences among type I IFNs did not explain their sub-classification, although differences at positions 94 through 109 and 175 through 189 were present between the signature and other IFNs. Conclusion Seven IFN-α isoforms and IFN-β participate in the late phase polarization of MPs conditioned by LPS. This information broadens the previous view of the central role played by IFN-α in

VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A

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Pio Ruben

2010-12-01

Full Text Available Abstract Background Different isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189 have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGFxxxb, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. Results Recombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGFxxxb isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p xxxb and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033 between VEGFxxxb and total VEGF-A was found. Conclusions Our results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGFxxxb isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken

Radiation-chemical synthesis of polypropylene fabrics with sulfonic acid functional groups

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Cho, Hyun Kug; Park, Jung Soo; Han, Do Hung, E-mail: dhhan@yumail.ac.kr; Bondar, Iuliia, E-mail: juliavad@yahoo.co

2011-04-01

A sorption-active material carrying sulfonic acid groups was synthesized by the radiation-induced graft polymerization of styrene monomer onto the surface of non-woven polypropylene fabric, followed by sulfonation of the grafted polystyrene chains. The effect of the main experimental parameters (absorbed dose, monomer concentration, reaction time) on the styrene degree of grafting was investigated. The sulfonation process with 5% chlorosulfonic acid at room temperature was investigated in detail and the optimal sulfonation conditions for the samples with a medium degree of grafting (70-140%) were determined. Densities of 3.5-5 meq/g were obtained by applying those sorption-active PP fabrics with a sulfonic acid group.

N Termini of apPDE4 Isoforms Are Responsible for Targeting the Isoforms to Different Cellular Membranes

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Jang, Deok-Jin; Park, Soo-Won; Lee, Jin-A; Lee, Changhoon; Chae, Yeon-Su; Park, Hyungju; Kim, Min-Jeong; Choi, Sun-Lim; Lee, Nuribalhae; Kim, Hyoung; Kaang, Bong-Kiun

2010-01-01

Phosphodiesterases (PDEs) are known to play a key role in the compartmentalization of cAMP signaling; however, the molecular mechanisms underlying intracellular localization of different PDE isoforms are not understood. In this study, we have found that each of the supershort, short, and long forms of apPDE4 showed distinct localization in the…

Discovery of novel isoforms of huntingtin reveals a new hominid-specific exon.

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Albert Ruzo

Full Text Available Huntington's disease (HD is a devastating neurological disorder that is caused by an expansion of the poly-Q tract in exon 1 of the Huntingtin gene (HTT. HTT is an evolutionarily conserved and ubiquitously expressed protein that has been linked to a variety of functions including transcriptional regulation, mitochondrial function, and vesicle transport. This large protein has numerous caspase and calpain cleavage sites and can be decorated with several post-translational modifications such as phosphorylations, acetylations, sumoylations, and palmitoylations. However, the exact function of HTT and the role played by its modifications in the cell are still not well understood. Scrutiny of HTT function has been focused on a single, full length mRNA. In this study, we report the discovery of 5 novel HTT mRNA splice isoforms that are expressed in normal and HTT-expanded human embryonic stem cell (hESC lines as well as in cortical neurons differentiated from hESCs. Interestingly, none of the novel isoforms generates a truncated protein. Instead, 4 of the 5 new isoforms specifically eliminate domains and modifications to generate smaller HTT proteins. The fifth novel isoform incorporates a previously unreported additional exon, dubbed 41b, which is hominid-specific and introduces a potential phosphorylation site in the protein. The discovery of this hominid-specific isoform may shed light on human-specific pathogenic mechanisms of HTT, which could not be investigated with current mouse models of the disease.

Discovery of Novel Isoforms of Huntingtin Reveals a New Hominid-Specific Exon

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Popowski, Melissa; Haremaki, Tomomi; Croft, Gist F.; Deglincerti, Alessia; Brivanlou, Ali H.

2015-01-01

Huntington’s disease (HD) is a devastating neurological disorder that is caused by an expansion of the poly-Q tract in exon 1 of the Huntingtin gene (HTT). HTT is an evolutionarily conserved and ubiquitously expressed protein that has been linked to a variety of functions including transcriptional regulation, mitochondrial function, and vesicle transport. This large protein has numerous caspase and calpain cleavage sites and can be decorated with several post-translational modifications such as phosphorylations, acetylations, sumoylations, and palmitoylations. However, the exact function of HTT and the role played by its modifications in the cell are still not well understood. Scrutiny of HTT function has been focused on a single, full length mRNA. In this study, we report the discovery of 5 novel HTT mRNA splice isoforms that are expressed in normal and HTT-expanded human embryonic stem cell (hESC) lines as well as in cortical neurons differentiated from hESCs. Interestingly, none of the novel isoforms generates a truncated protein. Instead, 4 of the 5 new isoforms specifically eliminate domains and modifications to generate smaller HTT proteins. The fifth novel isoform incorporates a previously unreported additional exon, dubbed 41b, which is hominid-specific and introduces a potential phosphorylation site in the protein. The discovery of this hominid-specific isoform may shed light on human-specific pathogenic mechanisms of HTT, which could not be investigated with current mouse models of the disease. PMID:26010866

Differential expression of syndecan isoforms during mouse incisor amelogenesis.

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Muto, Taro; Miyoshi, Keiko; Munesue, Seiichi; Nakada, Hiroshi; Okayama, Minoru; Matsuo, Takashi; Noma, Takafumi

2007-08-01

Syndecans are transmembranous heparan sulfate proteoglycans (HSPGs) with covalently attached glycosaminoglycan side-chains located on the cell surface. The mammalian syndecan family is composed of four types of syndecans (syndecan-1 to -4). Syndecans interact with the intracellular cytoskeleton through the cytoplasmic domains of their core proteins and membrane proteins, extracellular enzymes, growth factors, and matrix components, through their heparan-sulfate chains, to regulate developmental processes.Here, as a first step to assess the possible roles of syndecan proteins in amelogenesis, we examined the expression patterns of all syndecan isoforms in continuously growing mouse incisors, in which we can overview major differentiation stages of amelogenesis at a glance. Understanding the expression domain of each syndecan isoform during specific developmental stages seems useful for investigating their physiological roles in amelogenesis. Immunohistochemical analysis of syndecan core proteins in the lower incisors from postnatal day 1 mice revealed spatially and temporally specific expression patterns, with syndecan-1 expressed in undifferentiated epithelial and mesenchymal cells, and syndecan-2, -3, and -4 in more differentiated cells. These findings suggest that each syndecan isoform functions distinctly during the amelogenesis of the incisors of mice.

Myosin isoform fiber type and fiber size in the tail of the Virginia opossum (Didelphis virginiana).

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Hazimihalis, P J; Gorvet, M A; Butcher, M T

2013-01-01

Muscle fiber type is a well studied property in limb muscles, however, much less is understood about myosin heavy chain (MHC) isoform expression in caudal muscles of mammalian tails. Didelphid marsupials are an interesting lineage in this context as all species have prehensile tails, but show a range of tail-function depending on either their arboreal or terrestrial locomotor habits. Differences in prehensility suggest that MHC isoform fiber types may also be different, in that terrestrial opossums may have a large distribution of oxidative fibers for object carrying tasks instead of faster, glycolytic fiber types expected in mammals with long tails. To test this hypothesis, MHC isoform fiber type and their regional distribution (proximal/transitional/distal) were determined in the tail of the Virginia opossum (Didelphis virginiana). Fiber types were determined by a combination of myosin-ATPase histochemistry, immunohistochemistry, and SDS-PAGE. Results indicate a predominance of the fast MHC-2A and -2X isoforms in each region of the tail. The presence of two fast isoforms, in addition to the slow MHC-1 isoform, was confirmed by SDS-PAGE analysis. The overall MHC isoform fiber type distribution for the tail was: 25% MHC-1, 71% MHC-2A/X hybrid, and 4% MHC-1/2A hybrid. Oxidative MHC-2A/X isoform fibers were found to be relatively large in cross-section compared to slow, oxidative MHC-1 and MHC-1/2A hybrid fibers. A large percentage of fast MHC-2A/X hybrids fibers may be suggestive of an evolutionary transition in MHC isoform distribution (fast-to-slow fiber type) in the tail musculature of an opossum with primarily a terrestrial locomotor habit and adaptive tail-function. Copyright © 2012 Wiley Periodicals, Inc.

SMRT has tissue-specific isoform profiles that include a form containing one CoRNR box

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Short, Stephen; Malartre, Marianne; Sharpe, Colin

2005-01-01

SMRT acts as a corepressor for a range of transcription factors. The amino-terminal part of the protein includes domains that mainly mediate transcriptional repression whilst the carboxy-terminal part includes domains that interact with nuclear receptors using up to three motifs called CoRNR boxes. The region of the SMRT primary transcript encoding the interaction domains is subject to alternative splicing that varies the inclusion of the third CoRNR box. The profile in mice includes an abundant, novel SMRT isoform that possesses just one CoRNR box. Mouse tissues therefore express SMRT isoforms containing one, two or three CoRNR boxes. In frogs, the SMRT isoform profile is tissue-specific. The mouse also shows distinct profiles generated by differential expression levels of the SMRT transcript isoforms. The formation of multiple SMRT isoforms and their tissue-specific regulation indicates a mechanism, whereby cells can define the repertoire of transcription factors regulated by SMRT

Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

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Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

1999-01-01

Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

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Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard [Laboratoire de Chimie Nucleaire Analytique et Bioenvironnementale, CNRS UMR5084, Universite Bordeaux 1, Chemin du Solarium, F-33175 Gradignan cedex (France); Solari, Pier Lorenzo [Synchrotron SOLEIL, L' Orme des Merisiers, BP 48, F-91192 Gif-sur-Yvette cedex, Saint-Aubin (France); Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis, E-mail: ortega@cenbg.in2p3.f [FAME, ESRF, 6 rue Jules Horowitz, BP220, F-38043 Grenoble cedex (France)

2009-11-15

Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

Science.gov (United States)

Chevreux, Sylviane; Solari, Pier Lorenzo; Roudeau, Stéphane; Deves, Guillaume; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis; Ortega, Richard

2009-11-01

Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

International Nuclear Information System (INIS)

Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard; Solari, Pier Lorenzo; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis

2009-01-01

Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

Gestational exposure to BDE-99 produces toxicity through upregulation of CYP isoforms and ROS production in the fetal rat liver.

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Blanco, Jordi; Mulero, Miquel; Domingo, José L; Sánchez, Domènec J

2012-05-01

On gestation day (GD) 6 to GD 19, pregnant Sprague Dawley rats were orally exposed to 0, 0.5, 1, and 2 mg/kg/day to one of the most prevalent polybrominated diphenyl ethers congeners found in humans, 2,2',4,4',5-pentaBDE (BDE-99). All dams were euthanized on GD 20, and live fetuses were evaluated for sex, body weight, and external, internal, and skeletal malformations and developmental variations. The liver from one fetus of each litter was excised for the evaluation of oxidative stress markers and the messenger RNA expression of multiple cytochrome P450 (CYP) isoforms. Exposure to BDE-99 during the gestational period produced delayed ossification, slight hypertrophy of the heart, and enlargement of the liver in fetuses. A transplacental effect of BDE-99, evidenced by the activation of nuclear hormones receptors that induce the upregulation of CYP1A1, CYP1A2, CYP2B1, and CYP3A2 isoforms, was also found in fetal liver. These isoforms are correlated with the activity level of the enzyme catalase and the levels of thiobarbituric acid reactive substances. However, teratogenic effects from BDE-99 exposure were not observed. Clear signs of embryo/fetal toxicity, due to a possible hormonal disruption, were evidenced by a large increase in the CYP system and the production of reactive oxygen species in fetal liver.

Skeletal muscle myosin heavy chain isoform content in relation to gonadal hormones and anabolic-catabolic balance in trained and untrained men.

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Grandys, Marcin; Majerczak, Joanna; Karasinski, Janusz; Kulpa, Jan; Zoladz, Jerzy A

2012-12-01

Gonadal hormones and anabolic-catabolic hormone balance have potent influence on skeletal muscle tissue, but little is known about their action with regard to myosin heavy chain (MHC) transformation in humans. We investigated the relationship between skeletal muscle MHC isoform content in the vastus lateralis muscle and basal testosterone (T) concentration in 3 groups of subjects: endurance trained (E), sprint/strength trained (S), and untrained (U) young men. We have also determined basal sex hormone-binding globulin and cortisol (C) concentrations in untrained subjects to examine the relationship between MHC composition and the anabolic-catabolic hormone balance. Moreover, basal free testosterone (fT) and bioavailable testosterone (bio-T) concentrations were calculated for this subgroup. Despite significant differences in MHC isoform content (69.4 ± 2.39%, 61.4 ± 8.04%, and 37.5 ± 13.80% of MHC-2 for groups S, U, and E, respectively, Kruskal-Wallis: H = 18.58, p 0.5). We have also found that in the U group, type 2 MHC in the vastus lateralis muscle is positively correlated with basal fT:C ratio (r = 0.63, p = 0.01). It is concluded that the differences in the training history and training specificity can be distinguished with regard to the MHC composition but not with regard to the basal T concentration. Simultaneously, it has been shown that MHC isoform content in human vastus lateralis muscle may be related to basal anabolic-catabolic hormone balance, and this hypothesis needs further investigation.

Arabidopsis RIBA Proteins: Two out of Three Isoforms Have Lost Their Bifunctional Activity in Riboflavin Biosynthesis

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Hiltunen, Hanna-Maija; Illarionov, Boris; Hedtke, Boris; Fischer, Markus; Grimm, Bernhard

2012-01-01

Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprised of GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS). Angiosperms share a small RIBA gene family consisting of three members. A reduction of AtRIBA1 expression in the Arabidopsis rfd1mutant and in RIBA1 antisense lines is not complemented by the simultaneously expressed isoforms AtRIBA2 and AtRIBA3. The intensity of the bleaching leaf phenotype of RIBA1 deficient plants correlates with the inactivation of AtRIBA1 expression, while no significant effects on the mRNA abundance of AtRIBA2 and AtRIBA3 were observed. We examined reasons why both isoforms fail to sufficiently compensate for a lack of RIBA1 expression. All three RIBA isoforms are shown to be translocated into chloroplasts as GFP fusion proteins. Interestingly, both AtRIBA2 and AtRIBA3 have amino acid exchanges in conserved peptides domains that have been found to be essential for the two enzymatic functions. In vitro activity assays of GCHII and DHBPS with all of the three purified recombinant AtRIBA proteins and complementation of E. coli ribA and ribB mutants lacking DHBPS and GCHII expression, respectively, confirmed the loss of bifunctionality for AtRIBA2 and AtRIBA3. Phylogenetic analyses imply that the monofunctional, bipartite RIBA3 proteins, which have lost DHBPS activity, evolved early in tracheophyte evolution. PMID:23203051

Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

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Ivanna Ihnatovych

Full Text Available Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C. Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate- cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

VEGF-A isoform-specific regulation of calcium ion flux, transcriptional activation and endothelial cell migration.

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Fearnley, Gareth W; Bruns, Alexander F; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

2015-04-24

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Numerous isoforms of VEGF-A exist but their physiological significance is unclear. Here we evaluated two different VEGF-A isoforms and discovered differential regulation of cytosolic calcium ion flux, transcription factor localisation and endothelial cell response. Analysis of VEGF-A isoform-specific stimulation of VEGFR2-dependent signal transduction revealed differential capabilities for isoform activation of multiple signal transduction pathways. VEGF-A165 treatment promoted increased phospholipase Cγ1 phosphorylation, which was proportional to the subsequent rise in cytosolic calcium ions, in comparison to cells treated with VEGF-A121. A major consequence of this VEGF-A isoform-specific calcium ion flux in endothelial cells is differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2. Using reverse genetics, we discovered that NFATc2 is functionally required for VEGF-A-stimulated endothelial cell migration but not tubulogenesis. This work presents a new mechanism for understanding how VEGF-A isoforms program complex cellular outputs by converting signal transduction pathways into transcription factor redistribution to the nucleus, as well as defining a novel role for NFATc2 in regulating the endothelial cell response. © 2015. Published by The Company of Biologists Ltd.

VEGF-A isoform-specific regulation of calcium ion flux, transcriptional activation and endothelial cell migration

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Gareth W. Fearnley

2015-07-01

Full Text Available Vascular endothelial growth factor A (VEGF-A regulates many aspects of vascular physiology such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Numerous isoforms of VEGF-A exist but their physiological significance is unclear. Here we evaluated two different VEGF-A isoforms and discovered differential regulation of cytosolic calcium ion flux, transcription factor localisation and endothelial cell response. Analysis of VEGF-A isoform-specific stimulation of VEGFR2-dependent signal transduction revealed differential capabilities for isoform activation of multiple signal transduction pathways. VEGF-A165 treatment promoted increased phospholipase Cγ1 phosphorylation, which was proportional to the subsequent rise in cytosolic calcium ions, in comparison to cells treated with VEGF-A121. A major consequence of this VEGF-A isoform-specific calcium ion flux in endothelial cells is differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2. Using reverse genetics, we discovered that NFATc2 is functionally required for VEGF-A-stimulated endothelial cell migration but not tubulogenesis. This work presents a new mechanism for understanding how VEGF-A isoforms program complex cellular outputs by converting signal transduction pathways into transcription factor redistribution to the nucleus, as well as defining a novel role for NFATc2 in regulating the endothelial cell response.

TANK-Binding Kinase 1 (TBK1 Isoforms Negatively Regulate Type I Interferon Induction by Inhibiting TBK1-IRF3 Interaction and IRF3 Phosphorylation

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Yi Wei Hu

2018-01-01

Full Text Available TANK-binding kinase 1 (TBK1 is an important serine/threonine-protein kinase that mediates phosphorylation and nuclear translocation of IRF3, which contributes to induction of type I interferons (IFNs in the innate antiviral response. In mammals, TBK1 spliced isoform negatively regulates the virus-triggered IFN-β signaling pathway by disrupting the interaction between retinoic acid-inducible gene I (RIG-I and mitochondria antiviral-signaling protein (MAVS. However, it is still unclear whether alternative splicing patterns and the function of TBK1 isoform(s exist in teleost fish. In this study, we identify two alternatively spliced isoforms of TBK1 from zebrafish, termed TBK1_tv1 and TBK1_tv2. Both TBK1_tv1 and TBK1_tv2 contain an incomplete STKc_TBK1 domain. Moreover, the UBL_TBK1_like domain is also missing for TBK1_tv2. TBK1_tv1 and TBK1_tv2 are expressed in zebrafish larvae. Overexpression of TBK1_tv1 and TBK1_tv2 inhibits RIG-I-, MAVS-, TBK1-, and IRF3-mediated activation of IFN promoters in response to spring viremia of carp virus infection. Also, TBK1_tv1 and TBK1_tv2 inhibit expression of IFNs and IFN-stimulated genes induced by MAVS and TBK1. Mechanistically, TBK1_tv1 and TBK1_tv2 competitively associate with TBK1 and IRF3 to disrupt the formation of a functional TBK1-IRF3 complex, impeding the phosphorylation of IRF3 mediated by TBK1. Collectively, these results demonstrate that TBK1 spliced isoforms are dominant negative regulators in the RIG-I/MAVS/TBK1/IRF3 antiviral pathway by targeting the functional TBK1-IRF3 complex formation. Identification and functional characterization of piscine TBK1 spliced isoforms may contribute to understanding the role of TBK1 expression in innate antiviral response.

Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

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Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

2015-04-03

The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

MAPA distinguishes genotype-specific variability of highly similar regulatory protein isoforms in potato tuber.

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Hoehenwarter, Wolfgang; Larhlimi, Abdelhalim; Hummel, Jan; Egelhofer, Volker; Selbig, Joachim; van Dongen, Joost T; Wienkoop, Stefanie; Weckwerth, Wolfram

2011-07-01

Mass Accuracy Precursor Alignment is a fast and flexible method for comparative proteome analysis that allows the comparison of unprecedented numbers of shotgun proteomics analyses on a personal computer in a matter of hours. We compared 183 LC-MS analyses and more than 2 million MS/MS spectra and could define and separate the proteomic phenotypes of field grown tubers of 12 tetraploid cultivars of the crop plant Solanum tuberosum. Protein isoforms of patatin as well as other major gene families such as lipoxygenase and cysteine protease inhibitor that regulate tuber development were found to be the primary source of variability between the cultivars. This suggests that differentially expressed protein isoforms modulate genotype specific tuber development and the plant phenotype. We properly assigned the measured abundance of tryptic peptides to different protein isoforms that share extensive stretches of primary structure and thus inferred their abundance. Peptides unique to different protein isoforms were used to classify the remaining peptides assigned to the entire subset of isoforms based on a common abundance profile using multivariate statistical procedures. We identified nearly 4000 proteins which we used for quantitative functional annotation making this the most extensive study of the tuber proteome to date.

Mechanisms of isoform-specific Na/K pump regulation by short- and long-term adrenergic activation in rat ventricular myocytes.

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Yin, Jian; Guo, Hui-Cai; Yu, Ding; Wang, Hui-Ci; Li, Jun-Xia; Wang, Yong-Li

2014-01-01

Many stressful conditions, including cardiovascular diseases, induce long-term elevations in circulating catecholamines, thereby leading to changes of the Na/K pump and thus affecting myocardial functions. However, only short-term adrenergic regulation of the Na/K pump has been reported. The present study is the first investigation of long-term adrenergic regulation of the Na/K pump and the potential mechanism. After acutely isolated Sprague-Dawley rat myocytes were incubated with noradrenaline or isoprenaline for 24 h, Na/K pump high- (IPH) and low-affinity current (IPL), α-isoform mRNA, and α-isoform protein were examined using patch-clamp, RT-PCR, and Western blotting techniques, respectively. After the short-term incubation, isoprenaline reduced the IPL through a PKA-dependent pathway that involves α1-isoform translocation from the membrane to early endosomes, and noradrenaline increased the IPH through a PKC-dependent pathway that involves α2-isoform translocation from late endosomes to the membrane. After long-term incubation, isoprenaline increased the IPL, α1-isoform mRNA, and α1-isoform protein, and noradrenaline reduced the IPH, α2-isoform mRNA, and α1-isoform protein through a PKA-or PKC-dependent pathway, respectively. These results suggest that long-term adrenergic Na/K pump regulation is isoform-specific and negatively feeds back on the short-term response. Furthermore, long-term regulation involves transcription and translation of the respective α-isoform, whereas short-term regulation involves the translocation of the available α-isoform to the plasma membrane. © 2014 S. Karger AG, Basel.

Mechanisms of Isoform-Specific Na/K Pump Regulation by Short- and Long-Term Adrenergic Activation in Rat Ventricular Myocytes

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Jian Yin

2014-05-01

Full Text Available Background: Many stressful conditions, including cardiovascular diseases, induce long-term elevations in circulating catecholamines, thereby leading to changes of the Na/K pump and thus affecting myocardial functions. However, only short-term adrenergic regulation of the Na/K pump has been reported. The present study is the first investigation of long-term adrenergic regulation of the Na/K pump and the potential mechanism. Methods: After acutely isolated Sprague-Dawley rat myocytes were incubated with noradrenaline or isoprenaline for 24 h, Na/K pump high- (IPH and low-affinity current (IPL, α-isoform mRNA, and α-isoform protein were examined using patch-clamp, RT-PCR, and Western blotting techniques, respectively. Results: After the short-term incubation, isoprenaline reduced the IPL through a PKA-dependent pathway that involves α1-isoform translocation from the membrane to early endosomes, and noradrenaline increased the IPH through a PKC-dependent pathway that involves α2-isoform translocation from late endosomes to the membrane. After long-term incubation, isoprenaline increased the IPL, α1-isoform mRNA, and α1-isoform protein, and noradrenaline reduced the IPH, α2-isoform mRNA, and α1-isoform protein through a PKA-or PKC-dependent pathway, respectively. Conclusions: These results suggest that long-term adrenergic Na/K pump regulation is isoform-specific and negatively feeds back on the short-term response. Furthermore, long-term regulation involves transcription and translation of the respective α-isoform, whereas short-term regulation involves the translocation of the available α-isoform to the plasma membrane.

The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease.

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Clausen, Michael V; Hilbers, Florian; Poulsen, Hanne

2017-01-01

The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

Crystallization and preliminary X-ray diffraction studies of NP24-I, an isoform of a thaumatin-like protein from ripe tomato fruits

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Ghosh, Raka; Chakrabarti, Chandana, E-mail: chandana.chakrabarti@saha.ac.in [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata 700064 (India)

2005-08-01

A thaumatin-like antifungal protein, NP24-I, has been isolated from ripe tomato fruits. It was crystallized by the vapour-diffusion method and data were collected to 2.45 Å. The structure was solved by molecular replacement. NP24 is a 24 kDa (207-amino-acid) antifungal thaumatin-like protein (TLP) found in tomato fruits. An isoform of the protein, NP24-I, is reported to play a possible role in ripening of the fruit in addition to its antifungal properties. The protein has been isolated and purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the tetragonal space group P4{sub 3}, with unit-cell parameters a = b = 61.01, c = 62.90 Å and one molecule per asymmetric unit. X-ray diffraction data were processed to a resolution of 2.45 Å and the structure was solved by molecular replacement.

Acid Stress Response Mechanisms of Group B Streptococci

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Sarah Shabayek

2017-09-01

Full Text Available Group B streptococcus (GBS is a leading cause of neonatal mortality and morbidity in the United States and Europe. It is part of the vaginal microbiota in up to 30% of pregnant women and can be passed on to the newborn through perinatal transmission. GBS has the ability to survive in multiple different host niches. The pathophysiology of this bacterium reveals an outstanding ability to withstand varying pH fluctuations of the surrounding environments inside the human host. GBS host pathogen interations include colonization of the acidic vaginal mucosa, invasion of the neutral human blood or amniotic fluid, breaching of the blood brain barrier as well as survival within the acidic phagolysosomal compartment of macrophages. However, investigations on GBS responses to acid stress are limited. Technologies, such as whole genome sequencing, genome-wide transcription and proteome mapping facilitate large scale identification of genes and proteins. Mechanisms enabling GBS to cope with acid stress have mainly been studied through these techniques and are summarized in the current review

Statistical modeling of isoform splicing dynamics from RNA-seq time series data.

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Huang, Yuanhua; Sanguinetti, Guido

2016-10-01

Isoform quantification is an important goal of RNA-seq experiments, yet it remains problematic for genes with low expression or several isoforms. These difficulties may in principle be ameliorated by exploiting correlated experimental designs, such as time series or dosage response experiments. Time series RNA-seq experiments, in particular, are becoming increasingly popular, yet there are no methods that explicitly leverage the experimental design to improve isoform quantification. Here, we present DICEseq, the first isoform quantification method tailored to correlated RNA-seq experiments. DICEseq explicitly models the correlations between different RNA-seq experiments to aid the quantification of isoforms across experiments. Numerical experiments on simulated datasets show that DICEseq yields more accurate results than state-of-the-art methods, an advantage that can become considerable at low coverage levels. On real datasets, our results show that DICEseq provides substantially more reproducible and robust quantifications, increasing the correlation of estimates from replicate datasets by up to 10% on genes with low or moderate expression levels (bottom third of all genes). Furthermore, DICEseq permits to quantify the trade-off between temporal sampling of RNA and depth of sequencing, frequently an important choice when planning experiments. Our results have strong implications for the design of RNA-seq experiments, and offer a novel tool for improved analysis of such datasets. Python code is freely available at http://diceseq.sf.net G.Sanguinetti@ed.ac.uk Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

P1 promoter-driven HNF4α isoforms are specifically repressed by β-catenin signaling in colorectal cancer cells.

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Babeu, Jean-Philippe; Jones, Christine; Geha, Sameh; Carrier, Julie C; Boudreau, François

2018-06-13

HNF4α is a key nuclear receptor for regulating gene expression in the gut. While both P1 and P2 isoform classes of HNF4α are expressed in colonic epithelium, specific inhibition of P1 isoforms is commonly found in colorectal cancer. Previous studies have suggested that P1 and P2 isoforms may regulate different cellular functions. Despite these advances, it remains unclear whether these isoform classes are functionally divergent in the context of human biology. Here, the consequences of specific inhibition of P1 or P2 isoform expression was measured in a human colorectal cancer cell transcriptome. Results indicate that P1 isoforms were specifically associated with the control of cell metabolism while P2 isoforms globally supported aberrant oncogenic signalization, promoting cancer cell survival and progression. P1 promoter-driven isoform expression was found to be repressed by β-catenin, one of the earliest oncogenic pathways to be activated during colon tumorigenesis. These findings identify a novel cascade by which the expression of P1 isoforms are rapidly shut down in the early stages of colon tumorigenesis, allowing a change in HNF4α-dependent transcriptome thereby promoting colorectal cancer progression. © 2018. Published by The Company of Biologists Ltd.

[Correlation Between Functional Groups and Radical Scavenging Activities of Acidic Polysaccharides from Dendrobium].

Science.gov (United States)

Liao, Ying; Yuan, Wen-yu; Zheng, Wen-ke; Luo, Ao-xue; Fan, Yi-jun

2015-11-01

To compare the radical scavenging activity of five different acidic polysaccharides, and to find the correlation with the functional groups. Alkali extraction method and Stepwise ethanol precipitation method were used to extract and concentrate the five Dendrobium polysaccharides, and to determine the contents of sulfuric acid and uronic acid of each kind of acidic polysaccharides, and the scavenging activity to ABTS+ radical and hydroxyl radical. Functional group structures were examined by FTIR Spectrometer. Five kinds of Dendrobium polysaccharides had different ability of scavenging ABTS+ free radical and hydroxyl free radical. Moreover, the study had shown that five kinds of antioxidant activity of acidic polysaccharides had obvious correlation withuronic acid and sulfuric acid. The antioxidant activity of each sample was positively correlated with the content of uronic acid, and negatively correlated with the content of sulfuric acid. Sulfuric acid can inhibit the antioxidant activity of acidic polysaccharide but uronic acid can enhance the free radical scavenging activity. By analyzing the structure characteristics of five acidic polysaccharides, all samples have similar structures, however, Dendrobium denneanum, Dendrobium devonianum and Dendrobium officinale which had β configuration have higher antioxidant activity than Dendrobium nobile and Dendrobium fimbriatum which had a configuration.

High molecular weight FGF2 isoforms demonstrate canonical receptor-mediated activity and support human embryonic stem cell self-renewal

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Denis Kole

2017-05-01

Full Text Available Basic fibroblast growth factor (FGF2 is a highly pleiotropic member of a large family of growth factors with a broad range of activities, including mitogenesis and angiogenesis (Ornitz et al., 1996; Zhang et al., 2006, and it is known to be essential for maintenance of balance between survival, proliferation, and self-renewal in human pluripotent stem cells (Eiselleova et al., 2009; Zoumaro-Djayoon et al., 2011. A single FGF2 transcript can be translated into five FGF2 protein isoforms, an 18 kDa low molecular weight (LMW isoform and four larger high molecular weight (HMW isoforms (Arese et al., 1999; Arnaud et al., 1999. As they are not generally secreted, high molecular weight (HMW FGF2 isoforms have predominantly been investigated intracellularly; only a very limited number of studies have investigated their activity as extracellular factors. Here we report over-expression, isolation, and biological activity of all recombinant human FGF2 isoforms. We show that HMW FGF2 isoforms can support self-renewal of human embryonic stem cells (hESCs in vitro. Exogenous supplementation with HMW FGF2 isoforms also activates the canonical FGFR/MAPK pathway and induces mitogenic activity in a manner similar to that of the 18 kDa FGF2 isoform. Though all HMW isoforms, when supplemented exogenously, are able to recapitulate LMW FGF2 activity to some degree, it appears that certain isoforms tend to do so more poorly, demonstrating a lesser functional response by several measures. A better understanding of isoform-specific FGF2 effects will lead to a better understanding of developmental and pathological FGF2 signaling.

The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease

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Michael V. Clausen

2017-06-01

Full Text Available The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation

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Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K.; Klein, Gerd

2015-01-01

Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells. PMID:26406476

Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation.

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Tanja Seeger

Full Text Available Multipotent mesenchymal stromal cells (MSCs are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521 showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells.

Xcat, a novel mouse model for Nance-Horan syndrome inhibits expression of the cytoplasmic-targeted Nhs1 isoform.

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Huang, Kristen M; Wu, Junhua; Duncan, Melinda K; Moy, Chris; Dutra, Amalia; Favor, Jack; Da, Tong; Stambolian, Dwight

2006-01-15

Nance-Horan syndrome (NHS) is an X-linked disorder characterized by congenital cataracts, dental anomalies, dysmorphic features and mental retardation. A recent report suggests that the novel gene NHS1 is involved in this disorder due to the presence of point mutations in NHS patients. A possible mouse model for NHS, Xcat, was mapped to a 2.11 Mb interval on the X-chromosome. Sequence and FISH analysis of the X-chromosome region containing the Xcat mutation reveal a large insertion between exons 1 and 2 of the mouse Nhs1 gene. The insertion inhibits the expression of the Nhs1 isoform containing exon 1 and results in exclusive expression of the alternative isoform containing exon 1A. Quantitative RT-PCR of Xcat cDNA shows reduced levels of Nhs1 transcripts. The Nhs1 protein is strongly expressed within the cytoplasm of elongating lens fiber cells from wild-type neonate lens, but is significantly reduced within the Xcat lens. Transient transfection studies of CHO cells with Nhs1-GFP fusion proteins were done to determine whether the amino acids encoded by exon 1 were critical for protein localization. We found the presence of Nhs1 exon 1 critical for localization of the fusion protein to the cytoplasm, whereas fusion proteins lacking Nhs1 exon 1 are predominantly nuclear. These results indicate that the first exon of Nhs1 contains crucial information required for the proper expression and localization of Nhs1 protein. Inhibition of expression of the exon 1 containing isoform results in the abnormal phenotype of Xcat.

Brain region-specific expression of MeCP2 isoforms correlates with DNA methylation within Mecp2 regulatory elements.

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Carl O Olson

Full Text Available MeCP2 is a critical epigenetic regulator in brain and its abnormal expression or compromised function leads to a spectrum of neurological disorders including Rett Syndrome and autism. Altered expression of the two MeCP2 isoforms, MeCP2E1 and MeCP2E2 has been implicated in neurological complications. However, expression, regulation and functions of the two isoforms are largely uncharacterized. Previously, we showed the role of MeCP2E1 in neuronal maturation and reported MeCP2E1 as the major protein isoform in the adult mouse brain, embryonic neurons and astrocytes. Recently, we showed that DNA methylation at the regulatory elements (REs within the Mecp2 promoter and intron 1 impact the expression of Mecp2 isoforms in differentiating neural stem cells. This current study is aimed for a comparative analysis of temporal, regional and cell type-specific expression of MeCP2 isoforms in the developing and adult mouse brain. MeCP2E2 displayed a later expression onset than MeCP2E1 during mouse brain development. In the adult female and male brain hippocampus, both MeCP2 isoforms were detected in neurons, astrocytes and oligodendrocytes. Furthermore, MeCP2E1 expression was relatively uniform in different brain regions (olfactory bulb, striatum, cortex, hippocampus, thalamus, brainstem and cerebellum, whereas MeCP2E2 showed differential enrichment in these brain regions. Both MeCP2 isoforms showed relatively similar distribution in these brain regions, except for cerebellum. Lastly, a preferential correlation was observed between DNA methylation at specific CpG dinucleotides within the REs and Mecp2 isoform-specific expression in these brain regions. Taken together, we show that MeCP2 isoforms display differential expression patterns during brain development and in adult mouse brain regions. DNA methylation patterns at the Mecp2 REs may impact this differential expression of Mecp2/MeCP2 isoforms in brain regions. Our results significantly contribute

Synthesis, characterization and catalytic activity of acid-base bifunctional materials through protection of amino groups

Energy Technology Data Exchange (ETDEWEB)

Shao, Yanqiu [College of Chemistry, Jilin University, Changchun 130023 (China); College of Chemistry, Mudanjiang Normal University, Mudanjiang 157012 (China); Liu, Heng; Yu, Xiaofang [College of Chemistry, Jilin University, Changchun 130023 (China); Guan, Jingqi, E-mail: guanjq@jlu.edu.cn [College of Chemistry, Jilin University, Changchun 130023 (China); Kan, Qiubin, E-mail: qkan@mail.jlu.edu.cn [College of Chemistry, Jilin University, Changchun 130023 (China)

2012-03-15

Graphical abstract: Acid-base bifunctional mesoporous material SO{sub 3}H-SBA-15-NH{sub 2} was successfully synthesized under low acidic medium through protection of amino groups. Highlights: Black-Right-Pointing-Pointer The acid-base bifunctional material SO{sub 3}H-SBA-15-NH{sub 2} was successfully synthesized through protection of amino groups. Black-Right-Pointing-Pointer The obtained bifunctional material was tested for aldol condensation. Black-Right-Pointing-Pointer The SO{sub 3}H-SBA-15-NH{sub 2} catalyst containing amine and sulfonic acid groups exhibited excellent acid-basic properties. -- Abstract: Acid-base bifunctional mesoporous material SO{sub 3}H-SBA-15-NH{sub 2} was successfully synthesized under low acidic medium through protection of amino groups. X-ray diffraction (XRD), N{sub 2} adsorption-desorption, transmission electron micrographs (TEM), back titration, {sup 13}C magic-angle spinning (MAS) NMR and {sup 29}Si magic-angle spinning (MAS) NMR were employed to characterize the synthesized materials. The obtained bifunctional material was tested for aldol condensation reaction between acetone and 4-nitrobenzaldehyde. Compared with monofunctional catalysts of SO{sub 3}H-SBA-15 and SBA-15-NH{sub 2}, the bifunctional sample of SO{sub 3}H-SBA-15-NH{sub 2} containing amine and sulfonic acid groups exhibited excellent acid-basic properties, which make it possess high activity for the aldol condensation.

Effect of renal replacement therapy on retinol-binding protein 4 isoforms

DEFF Research Database (Denmark)

Frey, Simone K; Henze, Andrea; Nagl, Britta

2009-01-01

Retinol-binding protein 4 (RBP4) levels are elevated in the serum of patients with kidney dysfunction. We recently showed that RBP4 isoforms including apo-RBP4 (RBP4 not bound to retinol) and RBP4 truncated at the C-terminus (RBP4-L, RBP4-LL) are increased in the serum of patients with kidney dis...... diseases but not in serum of patients with various liver diseases. The aim of this study was to investigate the effect of renal replacement therapy on RBP4 isoforms....

Isoforms of transferrin in psoriasis patients abusing alcohol

NARCIS (Netherlands)

P. Hoefkens (Peter); E.M. Higgins; R.J. Ward (Roberta); H.G. van Eijk (Henk)

1997-01-01

textabstractThe different isoforms of transferrin have been quantified by isoelectric focusing in the sera of psoriasis patients with and without a history of abusing alcohol. In both male and female psoriasis subjects abusing alcohol, there were significant increases in the

Progesterone receptor isoform A may regulate the effects of neoadjuvant aglepristone in canine mammary carcinoma

DEFF Research Database (Denmark)

Guil-Luna, Silvia; Stenvang, Jan; Brünner, Nils

2014-01-01

RNA expression of progesterone receptor isoforms A and B in mammary carcinomas in dogs treated with 20 mg/Kg of aglepristone (n¿=¿22) or vehicle (n¿=¿5) twice before surgery.ResultsFormalin-fixed, paraffin-embedded tissue samples taken before and after treatment were used to analyse total progesterone receptor......-receptor positive and isoform-A positive tumours in aglepristone-treated dogs.ConclusionsThese findings suggest that the antiproliferative effects of aglepristone in canine mammary carcinomas are mediated by progesterone receptor isoform A....

Population pharmacokinetics of modafinil acid and estimation of the metabolic conversion of modafinil into modafinil acid in 5 major ethnic groups of China.

Science.gov (United States)

Wu, Ke-hua; Guo, Tao; Deng, Chen-hui; Guan, Zheng; Li, Liang; Zhou, Tian-yan; Lu, Wei

2012-11-01

To describe the population pharmacokinetic profile of modafinil acid and to compare the extent of metabolism of modafinil into modafinil acid in 5 major ethnic groups (Han, Mongolian, Korean, Uygur, and Hui) of China. In a multi-center, open-label, single dose clinical trial, 49 healthy volunteers from the 5 ethnic groups received 200 mg of modafinil orally. Blood samples for pharmacokinetic evaluation of modafinil and modafinil acid were drawn before and at different time after the administration. Systematic population pharmacokinetic (PopPK) modeling for modafinil acid was conducted, integrating with our previous PopPK model for modafinil. The influence of ethnicity, gender, height, body weight and body mass index (BMI) was estimated. The extent of metabolism of modafinil into modafinil acid, expressed as the relative conversion fraction, was estimated and compared among the 5 ethnic groups. When combined with the PopPK model of modafinil, the concentration of modafinil acid versus time profile was best described with a one-compartment model. The typical clearance and volume of distribution for modafinil acid were 4.94 (l/h) and 2.73 (l), respectively. The Korean group had 25% higher clearance, and the Uygur and Hui groups had 12% higher clearance than the Han group. The median for the relative conversion fraction was 0.53 for Koreans, and 0.24 for the other 4 ethnicities. Ethnicity has significant influence on the clearance of modafinil acid. When patients in the 5 ethnic groups are administered drugs or prodrugs catalyzed by esterases and/or amidases, the variability in the extent of drug metabolism should be considered.

Binding mechanisms for histamine and agmatine ligands in plasmid deoxyribonucleic acid purifications.

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Sousa, Ângela; Pereira, Patrícia; Sousa, Fani; Queiroz, João A

2014-10-31

Histamine and agmatine amino acid derivatives were immobilized into monolithic disks, in order to combine the specificity and selectivity of the ligand with the high mass transfer and binding capacity offered by monolithic supports, to purify potential plasmid DNA biopharmaceuticals. Different elution strategies were explored by changing the type and salt concentration, as well as the pH, in order to understand the retention pattern of different plasmids isoforms The pVAX1-LacZ supercoiled isoform was isolated from a mixture of pDNA isoforms by using NaCl increasing stepwise gradient and also by ammonium sulfate decreasing stepwise gradient, in both histamine and agmatine monoliths. Acidic pH in the binding buffer mainly strengthened ionic interactions with both ligands in the presence of sodium chloride. Otherwise, for histamine ligand, pH values higher than 7 intensified hydrophobic interactions in the presence of ammonium sulfate. In addition, circular dichroism spectroscopy studies revealed that the binding and elution chromatographic conditions, such as the combination of high ionic strength with extreme pH values can reversibly influence the structural stability of the target nucleic acid. Therefore, ascending sodium chloride gradients with pH manipulation can be preferable chromatographic conditions to be explored in the purification of plasmid DNA biopharmaceuticals, in order to avoid the environmental impact of ammonium sulfate. Copyright © 2014. Published by Elsevier B.V.

Roles of different IRES-dependent FGF2 isoforms in the acquisition of the major aggressive features of human metastatic melanoma.

Science.gov (United States)

Andreucci, Elena; Bianchini, Francesca; Biagioni, Alessio; Del Rosso, Mario; Papucci, Laura; Schiavone, Nicola; Magnelli, Lucia

2017-01-01

Fibroblast growth factor 2 (FGF2) is involved in many physiological and pathological processes. Fgf2 deregulation contributes to the acquisition of malignant features of melanoma and other cancers. FGF2 is an alternative translation product expressed as five isoforms, a low-molecular-weight (18 KDa) and four high-molecular-weight (22, 22.5, 24, 34 KDa) isoforms, with different subcellular distributions. An internal ribosomal entry site (IRES) in its mRNA controls the translation of all the isoforms with the exception for the cap-dependent 34 KDa. The 18-KDa isoform has been extensively studied, while very few is known about the roles of high molecular weight isoforms. FGF2 is known to promote melanoma development and progression. To disclose the differential contribution of FGF2 isoforms in melanoma, we forced the expression of IRES-dependent low-molecular-weight (LMW, 18 KDa) and high-molecular-weight (HMW, 22, 22.5, 24 KDa) isoforms in a human metastatic melanoma cell line. This comparative study highlights that, while LMW isoform confers stem-like features to melanoma cells and promotes angiogenesis, HMW isoforms induce higher migratory ability and contribute to tumor perfusion by promoting vasculogenic mimicry (VM) when endothelial cell-driven angiogenesis is lacking. To conclude, FGF2 isoforms mainly behave in specific, antithetical manners, but can cooperate in different steps of tumor progression, providing melanoma cells with major malignant features. FGF2 is an alternative translation product expressed as different isoforms termed LMW and HMW. FGF2 is involved in melanoma development and progression. HMW FGF2 isoforms enhance in vitro motility of melanoma cells. LMW FGF2 confers stem-like features and increases in vivo metastasization. LMW FGF2 promotes angiogenesis while HMW FGF2 induces vasculogenic mimicry.

Muscle-Type Specific Autophosphorylation of CaMKII Isoforms after Paced Contractions

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Wouter Eilers

2014-01-01

Full Text Available We explored to what extent isoforms of the regulator of excitation-contraction and excitation-transcription coupling, calcium/calmodulin protein kinase II (CaMKII contribute to the specificity of myocellular calcium sensing between muscle types and whether concentration transients in its autophosphorylation can be simulated. CaMKII autophosphorylation at Thr287 was assessed in three muscle compartments of the rat after slow or fast motor unit-type stimulation and was compared against a computational model (CaMuZclE coupling myocellular calcium dynamics with CaMKII Thr287 phosphorylation. Qualitative differences existed between fast- (gastrocnemius medialis and slow-type muscle (soleus for the expression pattern of CaMKII isoforms. Phospho-Thr287 content of δA CaMKII, associated with nuclear functions, demonstrated a transient and compartment-specific increase after excitation, which contrasted to the delayed autophosphorylation of the sarcoplasmic reticulum-associated βM CaMKII. In soleus muscle, excitation-induced δA CaMKII autophosphorylation demonstrated frequency dependence (P = 0.02. In the glycolytic compartment of gastrocnemius medialis, CaMKII autophosphorylation after excitation was blunted. In silico assessment emphasized the importance of mitochondrial calcium buffer capacity for excitation-induced CaMKII autophosphorylation but did not predict its isoform specificity. The findings expose that CaMKII autophosphorylation with paced contractions is regulated in an isoform and muscle type-specific fashion and highlight properties emerging for phenotype-specific regulation of CaMKII.

Deregulation of the endogenous C/EBPβ LIP isoform predisposes to tumorigenesis.

Science.gov (United States)

Bégay, Valérie; Smink, Jeske J; Loddenkemper, Christoph; Zimmermann, Karin; Rudolph, Cornelia; Scheller, Marina; Steinemann, Doris; Leser, Ulf; Schlegelberger, Brigitte; Stein, Harald; Leutz, Achim

2015-01-01

Two long and one truncated isoforms (termed LAP*, LAP, and LIP, respectively) of the transcription factor CCAAT enhancer binding protein beta (C/EBPβ) are expressed from a single intronless Cebpb gene by alternative translation initiation. Isoform expression is sensitive to mammalian target of rapamycin (mTOR)-mediated activation of the translation initiation machinery and relayed through an upstream open reading frame (uORF) on the C/EBPβ mRNA. The truncated C/EBPβ LIP, initiated by high mTOR activity, has been implied in neoplasia, but it was never shown whether endogenous C/EBPβ LIP may function as an oncogene. In this study, we examined spontaneous tumor formation in C/EBPβ knockin mice that constitutively express only the C/EBPβ LIP isoform from its own locus. Our data show that deregulated C/EBPβ LIP predisposes to oncogenesis in many tissues. Gene expression profiling suggests that C/EBPβ LIP supports a pro-tumorigenic microenvironment, resistance to apoptosis, and alteration of cytokine/chemokine expression. The results imply that enhanced translation reinitiation of C/EBPβ LIP promotes tumorigenesis. Accordingly, pharmacological restriction of mTOR function might be a therapeutic option in tumorigenesis that involves enhanced expression of the truncated C/EBPβ LIP isoform. Elevated C/EBPβ LIP promotes cancer in mice. C/EBPβ LIP is upregulated in B-NHL. Deregulated C/EBPβ LIP alters apoptosis and cytokine/chemokine networks. Deregulated C/EBPβ LIP may support a pro-tumorigenic microenvironment.

Differential CARM1 Isoform Expression in Subcellular Compartments and among Malignant and Benign Breast Tumors.

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David Shlensky

Full Text Available Coactivator-associated arginine methyltransferase 1 (CARM1 is a coactivator for ERα and cancer-relevant transcription factors, and can methylate diverse cellular targets including histones. CARM1 is expressed in one of two alternative splice isoforms, full-length CARM1 (CARM1FL and truncated CARM1 (CARM1ΔE15. CARM1FL and CARM1ΔE15 function differently in transcriptional regulation, protein methylation, and mediation of pre-mRNA splicing in cellular models.To investigate the functional roles and the prognosis potential of CARM1 alternative spliced isoforms in breast cancer, we used recently developed antibodies to detect differential CARM1 isoform expression in subcellular compartments and among malignant and benign breast tumors.Immunofluorescence in MDA-MB-231 and BG-1 cell lines demonstrated that CARM1ΔE15 is the dominant isoform expressed in the cytoplasm, and CARM1FL is more nuclear localized. CARM1ΔE15 was found to be more sensitive to Hsp90 inhibition than CARM1FL, indicating that the truncated isoform may be the oncogenic form. Clinical cancer samples did not have significantly higher expression of CARM1FL or CARM1ΔE15 than benign breast samples at the level of mRNA or histology. Furthermore neither CARM1FL nor CARM1ΔE15 expression correlated with breast cancer molecular subtypes, tumor size, or lymph node involvement.The analysis presented here lends new insights into the possible oncogenic role of CARM1ΔE15. This study also demonstrates no obvious association of CARM1 isoform expression and clinical correlates in breast cancer. Recent studies, however, have shown that CARM1 expression correlates with poor prognosis, indicating a need for further studies of both CARM1 isoforms in a large cohort of breast cancer specimens.

Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents

OpenAIRE

Benoit, Stephen C.; Kemp, Christopher J.; Elias, Carol F.; Abplanalp, William; Herman, James P.; Migrenne, Stephanie; Lefevre, Anne-Laure; Cruciani-Guglielmacci, Céline; Magnan, Christophe; Yu, Fang; Niswender, Kevin; Irani, Boman G.; Holland, William L.; Clegg, Deborah J.

2009-01-01

Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-θ was expressed in discrete neuronal populations of ...

The Related Transcriptional Enhancer Factor-1 Isoform, TEAD4216, Can Repress Vascular Endothelial Growth Factor Expression in Mammalian Cells

Science.gov (United States)

Appukuttan, Binoy; McFarland, Trevor J.; Stempel, Andrew; Kassem, Jean B.; Hartzell, Matthew; Zhang, Yi; Bond, Derek; West, Kelsey; Wilson, Reid; Stout, Andrew; Pan, Yuzhen; Ilias, Hoda; Robertson, Kathryn; Klein, Michael L.; Wilson, David; Smith, Justine R.; Stout, J. Timothy

2012-01-01

Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4216, which represses VEGF promoter activity. The TEAD4216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4216 isoform can competitively repress the stimulatory activity of the TEAD4434 and TEAD4148 enhancers. Synthesis of the native VEGF165 protein and cellular proliferation is suppressed by the TEAD4216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases. PMID:22761647

The landscape of isoform switches in human cancers

DEFF Research Database (Denmark)

Vitting-Seerup, Kristoffer; Sandelin, Albin Gustav

2017-01-01

highly predictive of patient survival independent of cancer types. Our data constitute an important resource for cancer researchers, available through interactive web tools. Moreover, our methods, available as an R package, enable systematic analysis of isoform switches from other RNA-seq datasets...

VEGF(121)b, a new member of the VEGF(xxx)b family of VEGF-A splice isoforms, inhibits neovascularisation and tumour growth in vivo.

Science.gov (United States)

Rennel, E S; Varey, A H R; Churchill, A J; Wheatley, E R; Stewart, L; Mather, S; Bates, D O; Harper, S J

2009-10-06

The key mediator of new vessel formation in cancer and other diseases is VEGF-A. VEGF-A exists as alternatively spliced isoforms - the pro-angiogenic VEGF(xxx) family generated by exon 8 proximal splicing, and a sister family, termed VEGF(xxx)b, exemplified by VEGF(165)b, generated by distal splicing of exon 8. However, it is unknown whether this anti-angiogenic property of VEGF(165)b is a general property of the VEGF(xxx)b family of isoforms. The mRNA and protein expression of VEGF(121)b was studied in human tissue. The effect of VEGF(121)b was analysed by saturation binding to VEGF receptors, endothelial migration, apoptosis, xenograft tumour growth, pre-retinal neovascularisation and imaging of biodistribution in tumour-bearing mice with radioactive VEGF(121)b. The existence of VEGF(121)b was confirmed in normal human tissues. VEGF(121)b binds both VEGF receptors with similar affinity as other VEGF isoforms, but inhibits endothelial cell migration and is cytoprotective to endothelial cells through VEGFR-2 activation. Administration of VEGF(121)b normalised retinal vasculature by reducing both angiogenesis and ischaemia. VEGF(121)b reduced the growth of xenografted human colon tumours in association with reduced microvascular density, and an intravenous bolus of VEGF(121)b is taken up into colon tumour xenografts. Here we identify a second member of the family, VEGF(121)b, with similar properties to those of VEGF(165)b, and underline the importance of the six amino acids of exon 8b in the anti-angiogenic activity of the VEGF(xxx)b isoforms.

Functional divergence of platelet protein kinase C (PKC) isoforms in thrombus formation on collagen.

Science.gov (United States)

Gilio, Karen; Harper, Matthew T; Cosemans, Judith M E M; Konopatskaya, Olga; Munnix, Imke C A; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D; Heemskerk, Johan W M; Poole, Alastair W

2010-07-23

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent alpha-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCalpha and PKCbeta, whereas the novel isoform, PKC, negatively regulates these events. PKCdelta also negatively regulates thrombus formation but not alpha-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCalpha or PKCbeta showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKC. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen*

Science.gov (United States)

Gilio, Karen; Harper, Matthew T.; Cosemans, Judith M. E. M.; Konopatskaya, Olga; Munnix, Imke C. A.; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D.; Heemskerk, Johan W. M.; Poole, Alastair W.

2010-01-01

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen. PMID:20479008

A structured sparse regression method for estimating isoform expression level from multi-sample RNA-seq data.

Science.gov (United States)

Zhang, L; Liu, X J

2016-06-03

With the rapid development of next-generation high-throughput sequencing technology, RNA-seq has become a standard and important technique for transcriptome analysis. For multi-sample RNA-seq data, the existing expression estimation methods usually deal with each single-RNA-seq sample, and ignore that the read distributions are consistent across multiple samples. In the current study, we propose a structured sparse regression method, SSRSeq, to estimate isoform expression using multi-sample RNA-seq data. SSRSeq uses a non-parameter model to capture the general tendency of non-uniformity read distribution for all genes across multiple samples. Additionally, our method adds a structured sparse regularization, which not only incorporates the sparse specificity between a gene and its corresponding isoform expression levels, but also reduces the effects of noisy reads, especially for lowly expressed genes and isoforms. Four real datasets were used to evaluate our method on isoform expression estimation. Compared with other popular methods, SSRSeq reduced the variance between multiple samples, and produced more accurate isoform expression estimations, and thus more meaningful biological interpretations.

O-GlcNAcylation modulates PKA-CREB signaling in a manner specific to PKA catalytic subunit isoforms.

Science.gov (United States)

Jin, Nana; Ma, Denglei; Gu, Jianlan; Shi, Jianhua; Xu, Xiaotao; Iqbal, Khalid; Gong, Cheng-Xin; Liu, Fei; Chu, Dandan

2018-02-26

O-GlcNAcylation is a post-translational modification of proteins. Protein kinase A (PKA)-cAMP response element binding protein (CREB) signaling plays critical roles in multiple biological processes. Isoforms α and β of PKA catalytic subunit (PKAc) and CREB are modified by O-GlcNAcylation. In the present study, we determined the role of O-GlcNAcylation in PKAc isoform-specific CREB signaling. We found that up-regulation of O-GlcNAcylation enhanced CREB phosphorylation, but suppressed CREB expression in exogenous PKAc isoform-unspecific manner. PKAc isoforms affected exogenous expression of OGT or OGA and protein O-GlcNAcylation differently. Up-regulation of O-GlcNAcylation did not significantly affect net PKAcα-CREB signaling, but enhanced PKAcβ-CREB signaling. The role of O-GlcNAcylation in PKA-CREB signaling was desensitized by insulin treatment. This study suggests a role of O-GlcNAcylation in PKA-CREB signaling by affecting phosphorylation of CREB in a PKAc isoform-specific manner. Copyright © 2018 Elsevier Inc. All rights reserved.

Lipoprotein lipase isoelectric point isoforms in humans

DEFF Research Database (Denmark)

Badia-Villanueva, M.; Carulla, P.; Carrascal, M.

2014-01-01

-heparin plasma (PHP), LPL consists of a pattern of more than 8 forms of the same apparent molecular weight, but different isoelectric point (pI). In the present study we describe, for the first time, the existence of at least nine LPL pI isoforms in human PHP, with apparent pI between 6.8 and 8.6. Separation...

Myosin isoform switching during assembly of the Drosophila flight muscle thick filament lattice.

Science.gov (United States)

Orfanos, Zacharias; Sparrow, John C

2013-01-01

During muscle development myosin molecules form symmetrical thick filaments, which integrate with the thin filaments to produce the regular sarcomeric lattice. In Drosophila indirect flight muscles (IFMs) the details of this process can be studied using genetic approaches. The weeP26 transgenic line has a GFP-encoding exon inserted into the single Drosophila muscle myosin heavy chain gene, Mhc. The weeP26 IFM sarcomeres have a unique MHC-GFP-labelling pattern restricted to the sarcomere core, explained by non-translation of the GFP exon following alternative splicing. Characterisation of wild-type IFM MHC mRNA confirmed the presence of an alternately spliced isoform, expressed earlier than the major IFM-specific isoform. The two wild-type IFM-specific MHC isoforms differ by the presence of a C-terminal 'tailpiece' in the minor isoform. The sequential expression and assembly of these two MHCs into developing thick filaments suggest a role for the tailpiece in initiating A-band formation. The restriction of the MHC-GFP sarcomeric pattern in weeP26 is lifted when the IFM lack the IFM-specific myosin binding protein flightin, suggesting that it limits myosin dissociation from thick filaments. Studies of flightin binding to developing thick filaments reveal a progressive binding at the growing thick filament tips and in a retrograde direction to earlier assembled, proximal filament regions. We propose that this flightin binding restricts myosin molecule incorporation/dissociation during thick filament assembly and explains the location of the early MHC isoform pattern in the IFM A-band.

GSK3β isoform-selective regulation of depression, memory and hippocampal cell proliferation.

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Pardo, M; Abrial, E; Jope, R S; Beurel, E

2016-03-01

Abnormally active glycogen synthase kinase-3 (GSK3) contributes to pathological processes in multiple psychiatric and neurological disorders. Modeled in mice, this includes increasing susceptibility to dysregulation of mood-relevant behaviors, impairing performance in several cognitive tasks and impairing adult hippocampal neural precursor cell (NPC) proliferation. These deficits are all evident in GSK3α/β knockin mice, in which serine-to-alanine mutations block the inhibitory serine phosphorylation regulation of both GSK3 isoforms, leaving GSK3 hyperactive. It was unknown if both GSK3 isoforms perform redundant actions in these processes, or if hyperactivity of one GSK3 isoform has a predominant effect. To test this, we examined GSK3α or GSK3β knockin mice in which only one isoform was mutated to a hyperactive form. Only GSK3β, not GSK3α, knockin mice displayed heightened vulnerability to the learned helplessness model of depression-like behavior. Three cognitive measures impaired in GSK3α/β knockin mice showed differential regulation by GSK3 isoforms. Novel object recognition was impaired in GSK3β, not in GSK3α, knockin mice, whereas temporal order memory was not impaired in GSK3α or GSK3β knockin mice, and co-ordinate spatial processing was impaired in both GSK3α and GSK3β knockin mice. Adult hippocampal NPC proliferation was severely impaired in GSK3β knockin mice, but not impaired in GSK3α knockin mice. Increased activity of GSK3β, in the absence of overexpression or disease pathology, is sufficient to impair mood regulation, novel object recognition and hippocampal NPC proliferation, whereas hyperactive GSK3α individually does not impair these processes. These results show that hyperactivity of the two GSK3 isoforms execute non-redundant effects on these processes. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Tumour cells expressing single VEGF isoforms display distinct growth, survival and migration characteristics.

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Chryso Kanthou

Full Text Available Vascular endothelial growth factor-A (VEGF is produced by most cancer cells as multiple isoforms, which display distinct biological activities. VEGF plays an undisputed role in tumour growth, vascularisation and metastasis; nevertheless the functions of individual isoforms in these processes remain poorly understood. We investigated the effects of three main murine isoforms (VEGF188, 164 and 120 on tumour cell behaviour, using a panel of fibrosarcoma cells we developed that express them individually under endogenous promoter control. Fibrosarcomas expressing only VEGF188 (fs188 or wild type controls (fswt were typically mesenchymal, formed ruffles and displayed strong matrix-binding activity. VEGF164- and VEGF120-producing cells (fs164 and fs120 respectively were less typically mesenchymal, lacked ruffles but formed abundant cell-cell contacts. On 3D collagen, fs188 cells remained mesenchymal while fs164 and fs120 cells adopted rounded/amoeboid and a mix of rounded and elongated morphologies respectively. Consistent with their mesenchymal characteristics, fs188 cells migrated significantly faster than fs164 or fs120 cells on 2D surfaces while contractility inhibitors accelerated fs164 and fs120 cell migration. VEGF164/VEGF120 expression correlated with faster proliferation rates and lower levels of spontaneous apoptosis than VEGF188 expression. Nevertheless, VEGF188 was associated with constitutively active/phosphorylated AKT, ERK1/2 and Stat3 proteins. Differences in proliferation rates and apoptosis could be explained by defective signalling downstream of pAKT to FOXO and GSK3 in fs188 and fswt cells, which also correlated with p27/p21 cyclin-dependent kinase inhibitor over-expression. All cells expressed tyrosine kinase VEGF receptors, but these were not active/activatable suggesting that inherent differences between the cell lines are governed by endogenous VEGF isoform expression through complex interactions that are independent of tyrosine

Direct interaction of the mouse cytomegalovirus m152/gp40 immunoevasin with RAE-1 isoforms.

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Zhi, Li; Mans, Janet; Paskow, Michael J; Brown, Patrick H; Schuck, Peter; Jonjić, Stipan; Natarajan, Kannan; Margulies, David H

2010-03-23

Cytomegaloviruses (CMVs) are ubiquitous species-specific viruses that establish acute, persistent, and latent infections. Both human and mouse CMVs encode proteins that inhibit the activation of natural killer (NK) cells by downregulating cellular ligands for the NK cell activating receptor, NKG2D. The MCMV glycoprotein m152/gp40 downregulates the surface expression of RAE-1 to prevent NK cell control in vivo. So far, it is unclear if there is a direct interaction between m152 and RAE-1 and, if so, if m152 interacts differentially with the five identified RAE-1 isoforms, which are expressed as two groups in MCMV-susceptible or -resistant mouse strains. To address these questions, we expressed and purified the extracellular domains of RAE-1 and m152 and performed size exclusion chromatography binding assays as well as analytical ultracentrifugation and isothermal titration calorimetry to characterize these interactions quantitatively. We further evaluated the role of full-length and naturally glycosylated m152 and RAE-1 in cotransfected HEK293T cells. Our results confirmed that m152 binds RAE-1 directly, relatively tightly (K(d) RAE-1 isoforms, corresponding to the susceptibility to downregulation by m152. A PLWY motif found in RAE-1beta, although contributing to its affinity for m152, does not influence the affinity of RAE-1gamma or RAE-1delta, suggesting that other differences contribute to the RAE-1-m152 interaction. Molecular modeling of the different RAE-1 isoforms suggests a potential site for the m152 interaction.

Expression of two isoforms of CD44 in human endometrium.

Science.gov (United States)

Behzad, F; Seif, M W; Campbell, S; Aplin, J D

1994-10-01

The distribution of the cell-surface adhesion glycoprotein CD44 in human endometrium was examined by immunofluorescence using six monoclonal antibodies to epitopes common to all forms of the molecule, and by reverse transcription-polymerase chain reaction (RT-PCR). Immunoreactivity was observed throughout the menstrual cycle in stroma, vessels, glandular, and luminal epithelium. Variations in staining intensity were observed, especially in the epithelial compartment. CD44 was also expressed strongly by decidualized stromal cells of first-trimester pregnancy. No systematic variation of immunoreactivity was observed with stages of the normal cycle, but a fraction (25%) of the specimens lacked reactivity in the epithelium. To determine the molecular size of the epithelial isoform, an immunoprecipitation technique was developed using surface-radioiodinated, detergent-extracted glands. This indicated the presence at the cell surface of a single dominant CD44E species with an approximate molecular mass of 130 kDa. RT-PCR was used to investigate the isoforms present in whole endometrial tissue, isolated gland fragments, and Ishikawa endometrial carcinoma cells. Complementary DNA produced from total endometrial mRNA was PCR-amplified across the splice junction between exons 5 and 15. Transcripts corresponding to the hyaluronate receptor CD44H as well as a larger isoform were identified. CD44H was absent, or very scarce, in cDNA from purified gland epithelium. In contrast, Ishikawa cells expressed this form abundantly. The glands and Ishikawa cells also expressed CD44E containing sequences encoded by exons 12, 13, and 14. These data demonstrate the presence of CD44 in human endometrium and decidua, and show that different isoforms of CD44 are associated with tissue compartments in which different functional roles can be anticipated.

Smoking specifically induces metallothionein-2 isoform in human placenta at term

International Nuclear Information System (INIS)

Ronco, Ana Maria; Garrido, Fernando; Llanos, Miguel N.

2006-01-01

Recently, we reported the presence of higher levels of metallothionein (MT) in placentas of smokers compared to non-smokers. In the present study, we designed experiments to separate and evaluate two isoforms of MT (MT-1 and MT-2) in placentas of smokers and non-smokers. Metallothionein was extracted and separated by ion-exchange high performance liquid chromatography (HPLC), previous saturation with cadmium chloride. Two peaks eluting at 6 and 12.5 min, corresponding to MT-1 and MT-2, respectively, were obtained. Metallothionein present in both peaks was identified by Western blot analysis using a monoclonal antibody directed against MT-1 and MT-2. Each isoform concentration was calculated after measuring its cadmium content by atomic absorption spectrometry with inductively coupled-plasma. In placentas of smokers, MT-2 levels increased by seven-fold compared to non-smokers, whereas MT-1 was not changed. Total placental cadmium and zinc concentrations, determined by atomic absorption spectrometry and neutron activation analysis, respectively, were higher in smokers. Metallothioneins levels were clearly in excess to bind all cadmium ions present in placentas. However, most of placental zinc remains unbound to MTs, although as much as twice zinc ions could be bound to MT in smokers. In conclusion, MT-2 is the main isoform induced by smoking, suggesting that this isoform could be involved in placental cadmium and zinc retention. This fact, which could contribute to reduce the transference of zinc to the fetus, may be associated to detrimental effects on fetal growth and development

Nesprin-2 epsilon: A novel nesprin isoform expressed in human ovary and Ntera-2 cells

International Nuclear Information System (INIS)

Lam, Le Thanh; Boehm, Sabrina V.; Roberts, Roland G.; Morris, Glenn E.

2011-01-01

Highlights: → A novel epsilon isoform of nesprin-2 has been discovered. → This 120 kDa protein was predicted by bioinformatic analysis, but has not previously been observed. → It is the main isoform expressed in a teratocarcinoma cell line and is also found in ovary. → Like other nesprins, it is located at the nuclear envelope. → We suggest it may have a role in very early development or in some ovary-specific function. -- Abstract: The nuclear envelope-associated cytoskeletal protein, nesprin-2, is encoded by a large gene containing several internal promoters that produce shorter isoforms. In a study of Ntera-2 teratocarcinoma cells, a novel isoform, nesprin-2-epsilon, was found to be the major mRNA and protein product of the nesprin-2 gene. Its existence was predicted by bioinformatic analysis, but this is the first direct demonstration of both the mRNA and the 120 kDa protein which is located at the nuclear envelope. In a panel of 21 adult and foetal human tissues, the nesprin-2-epsilon mRNA was strongly expressed in ovary but was a minor isoform elsewhere. The expression pattern suggests a possible link with very early development and a likely physiological role in ovary.

2S protein Ara h 7.0201 has unique epitopes compared to other Ara h 7 isoforms and is comparable to 2S proteins Ara h 2 and 6 in basophil degranulation capacity.

Science.gov (United States)

Hayen, S M; Ehlers, A M; den Hartog Jager, C F; Garssen, J; Knol, E F; Knulst, A C; Suer, W; Willemsen, L E M; Otten, H G

2018-07-01

Screening for specific IgE against 2S albumin proteins Ara h 2 and 6 has good positive predictive value in diagnosing peanut allergy. From the third 2S member Ara h 7, 3 isoforms have been identified. Their allergenicity has not been elucidated. This study investigated the allergenicity of Ara h 7 isoforms compared to Ara h 2 and 6. Sensitization of 15 DBPCFC-confirmed peanut-allergic patients to recombinant Ara h 2.0201, Ara h 6.01 and isoforms of recombinant Ara h 7 was determined by IgE immunoblotting strips. A basophil activation test (BAT) was performed in 9 patients to determine IgE-cross-linking capacities of the allergens. Sensitivity to the allergens was tested in 5 patients who were sensitized to at least 1 Ara h 7 isoform, by a concentration range in the BAT. 3D prediction models and sequence alignments were used to visualize differences between isoforms and to predict allergenic epitope regions. Sensitization to Ara h 7.0201 was most frequent (80%) and showed to be equally potent as Ara h 2.0201 and 6.01 in inducing basophil degranulation. Sensitization to Ara h 7.0201 together with Ara h 2.0201 and/or 6.01 was observed, indicating the presence of unique epitopes compared to the other 2 isoforms. Differences between the 3 Ara h 7 isoforms were observed in C-terminal cysteine residues, pepsin and trypsin cleavage sites and 3 single amino acid substitutions. The majority of peanut-allergic patients are sensitized to isoform Ara h 7.0201, which is functionally as active as Ara h 2.0201 and 6.01. Unique epitopes are most likely located in the C-terminus or an allergenic loop region which is a known allergenic epitope region for Ara h 2.0201 and 6.01. Due to its unique epitopes and allergenicity, it is an interesting candidate to improve the diagnostic accuracy for peanut allergy. © 2018 The Authors. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd.

Effects of cavernous nerve reconstruction on expression of nitric oxide synthase isoforms in rats.

Science.gov (United States)

Schlenker, Boris; Matiasek, Kaspar; Saur, Dieter; Gratzke, Christian; Bauer, Ricarda M; Herouy, Yared; Arndt, Christian; Blesch, Armin; Hartung, Rudolf; Stief, Christian G; Weidner, Norbert; May, Florian

2010-12-01

To evaluate the expression of nitric oxide synthase (NOS) isoforms after various reconstruction techniques in rats, to improve the understanding of neuronal repair mechanisms after radical prostatectomy, as Schwann cell-seeded guidance tubes have been shown to promote cavernous nerve regeneration, and glial cell-line-derived neurotrophic factor (GDNF)-overexpressing Schwann cells enhance nerve regenerative capacity. Segments (5 mm) of the cavernous nerve were excised bilaterally, followed by immediate bilateral microsurgical reconstruction. In four rats per group, the eight nerves were reconstructed by autologous nerve grafting (A), interposition of Schwann cell-seeded silicon tubes (B), or silicon tubes seeded with GDNF-hypersecreting Schwann cells (C). Further rats were either sham-operated (D) or had nerve excision without repair (E). Erectile function was evaluated after 6 weeks by re-laparotomy, electrical nerve stimulation and morphological evaluation of reconstructed nerves. NOS isoform mRNA expression was analysed by reverse transcription-polymerase chain reaction in tissue specimens taken from the corpora cavernosa. GDNF-transduced Schwann cell grafts restored erectile function better than untransduced Schwann cell and autologous nerve grafts (88% vs 75% vs 38%; not significant). Tissue specimens in group C had the highest expression of neuronal NOS mRNA in relation to the neuronal marker PGP9.5 among all treatment groups (not significant). Compared to nerve grafts (A) and negative controls (E) nNOS/PGP9.5 expression was significantly higher (P Schwann cell grafts (P < 0.05). Restoration of erectile function is paralleled by an increase of neuronal NOS expression in rats. Further experiments will determine the physiological role of neuronal NOS in erectile nerve repair processes. © 2010 THE AUTHORS. JOURNAL COMPILATION © 2010 BJU INTERNATIONAL.

Uncovering the Rare Variants of DLC1 Isoform 1 and Their Functional Effects in a Chinese Sporadic Congenital Heart Disease Cohort

Science.gov (United States)

Wang, Zhen; Tan, Huilian; Kong, Xianghua; Shu, Yang; Zhang, Yuchao; Huang, Yun; Zhu, Yufei; Xu, Heng; Wang, Zhiqiang; Wang, Ping; Ning, Guang; Kong, Xiangyin; Hu, Guohong; Hu, Landian

2014-01-01

Congenital heart disease (CHD) is the most common birth defect affecting the structure and function of fetal hearts. Despite decades of extensive studies, the genetic mechanism of sporadic CHD remains obscure. Deleted in liver cancer 1 (DLC1) gene, encoding a GTPase-activating protein, is highly expressed in heart and essential for heart development according to the knowledge of Dlc1-deficient mice. To determine whether DLC1 is a susceptibility gene for sporadic CHD, we sequenced the coding region of DLC1 isoform 1 in 151 sporadic CHD patients and identified 13 non-synonymous rare variants (including 6 private variants) in the case cohort. Importantly, these rare variants (8/13) were enriched in the N-terminal region of the DLC1 isoform 1 protein. Seven of eight amino acids at the N-terminal variant positions were conserved among the primates. Among the 9 rare variants that were predicted as “damaging”, five were located at the N-terminal region. Ensuing in vitro functional assays showed that three private variants (Met360Lys, Glu418Lys and Asp554Val) impaired the ability of DLC1 to inhibit cell migration or altered the subcellular location of the protein compared to wild-type DLC1 isoform 1. These data suggest that DLC1 might act as a CHD-associated gene in addition to its role as a tumor suppressor in cancer. PMID:24587289

Molecular, cellular, and physiological responses to phosphatidic acid formation in plants

NARCIS (Netherlands)

Testerink, C.; Munnik, T.

2011-01-01

Phosphatidic acid (PA) is an essential phospholipid involved in membrane biosynthesis and signal transduction in all eukaryotes. This review focuses on its role as lipid second messenger during plant stress, metabolism, and development. The contribution of different individual isoforms of enzymes

Differential expression of a new isoform of DLG2 in renal oncocytoma

Directory of Open Access Journals (Sweden)

Kovacs Gyula

2006-04-01

Full Text Available Abstract Background Renal oncocytoma, a benign tumour of the kidney, may pose a differential diagnostic problem due to overlapping phenotype with chromophobe renal cell carcinoma or other types of renal cell tumours. Therefore, identification of molecular markers would be of great value for molecular diagnostics of this tumour type. Methods In the current study we applied various techniques, including Affymetrix microarray hybridization and semiquantitative RT-PCR, to identify genes expressed differentially in renal oncocytomas. Subsequently, we used RACE and Northern blot hybridization to characterize the potential candidates for molecular diagnosis. Results We have identified new isoform of DLG2 gene, which contains 3'-end exons of the known DLG2 gene along with the hypothetical gene FLJ37266. The new isoform is specifically upregulated in renal oncocytoma, whereas the known DLG2 gene is downregulated in this type of kidney tumour. Conclusion The new isoform of DLG2 is the promising candidate gene for molecular differential diagnostics of renal oncocytoma.

Differential expression of a new isoform of DLG2 in renal oncocytoma

International Nuclear Information System (INIS)

Zubakov, Dmitry; Stupar, Zorica; Kovacs, Gyula

2006-01-01

Renal oncocytoma, a benign tumour of the kidney, may pose a differential diagnostic problem due to overlapping phenotype with chromophobe renal cell carcinoma or other types of renal cell tumours. Therefore, identification of molecular markers would be of great value for molecular diagnostics of this tumour type. In the current study we applied various techniques, including Affymetrix microarray hybridization and semiquantitative RT-PCR, to identify genes expressed differentially in renal oncocytomas. Subsequently, we used RACE and Northern blot hybridization to characterize the potential candidates for molecular diagnosis. We have identified new isoform of DLG2 gene, which contains 3'-end exons of the known DLG2 gene along with the hypothetical gene FLJ37266. The new isoform is specifically upregulated in renal oncocytoma, whereas the known DLG2 gene is downregulated in this type of kidney tumour. The new isoform of DLG2 is the promising candidate gene for molecular differential diagnostics of renal oncocytoma

Kinetic characteristics of acidic and alkaline ceramidase in human epidermis

NARCIS (Netherlands)

Houben, E.; Uchida, Y.; Nieuwenhuizen, W.F.; Paepe, K. de; Vanhaecke, T.; Holleran, W.M.; Rogiers, V.

2007-01-01

It has recently become evident that at least five ceramidase (CDase) isoforms are present in human epidermis, and that specifically acidic CDase (aCDase) and alkaline CDase (alkCDase) activities increase during keratinocyte differentiation, and thus might play a pivotal role(s) in permeability

Two Distinct Isoforms of Matrix Metalloproteinase-2 Are Associated with Human Delayed Kidney Graft Function.

Directory of Open Access Journals (Sweden)

Shaynah Wanga

Full Text Available Delayed graft function (DGF is a frequent complication of renal transplantation, particularly in the setting of transplantation of kidneys derived from deceased donors and expanded-criteria donors. DGF results from tubular epithelial cell injury and has immediate and long term consequences. These include requirement for post-transplantation dialysis, increased incidence of acute rejection, and poorer long-term outcomes. DGF represents one of the clearest clinical examples of renal acute ischemia/reperfusion injury. Experimental studies have demonstrated that ischemia/reperfusion injury induces the synthesis of the full length secreted isoform of matrix metalloproteinase-2 (FL-MMP-2, as well as an intracellular N-terminal truncated MMP-2 isoform (NTT-MMP-2 that initiates an innate immune response. We hypothesized that the two MMP-2 isoforms mediate tubular epithelial cell injury in DGF. Archival renal biopsy sections from 10 protocol biopsy controls and 41 cases with a clinical diagnosis of DGF were analyzed for the extent of tubular injury, expression of the FL-MMP-2 and NTT-MMP-2 isoforms by immunohistochemistry (IHC, in situ hybridization, and qPCR to determine isoform abundance. Differences in transcript abundance were related to tubular injury score. Markers of MMP-2-mediated injury included TUNEL staining and assessment of peritubular capillary density. There was a clear relationship between tubular epithelial cell expression of both FL-MMP-2 and NTT-MMP-2 IHC with the extent of tubular injury. The MMP-2 isoforms were detected in the same tubular segments and were present at sites of tubular injury. qPCR demonstrated highly significant increases in both the FL-MMP-2 and NTT-MMP-2 transcripts. Statistical analysis revealed highly significant associations between FL-MMP-2 and NTT-MMP-2 transcript abundance and the extent of tubular injury, with NTT-MMP-2 having the strongest association. We conclude that two distinct MMP-2 isoforms are

Acid-Base Behavior of Carboxylic Acid Groups Covalently Attached at the Surface of Polyethylene: The Usefulness of Contact Angle in Following the Ionization of Surface Functionality

Science.gov (United States)

1985-08-01

additional check, we converted granular PE-CO 2H to granular PE-CO 2CH3 by acid -catalyzed esterification. This material had no titrable groups. Upon...Task No. NR-631-840 TECHNICAL REPORT NO. 85-1 Acid -Base Behavior of Carboxylic Acid Groups Covalently Attached at the Surface of Polyethylene: The...34I Acid -Base Behavior K-142 ofCarboxylicAcidGroupsAttached...______________________ 12. PERSIIMAL AUTHOR IS) S.R. Holmes-Farly., R.H. Reamey, T.J

Transcriptome-wide identification and characterization of CAD isoforms specific for podophyllotoxin biosynthesis from Podophyllum hexandrum.

Science.gov (United States)

Bhattacharyya, Dipto; Hazra, Saptarshi; Banerjee, Anindyajit; Datta, Riddhi; Kumar, Deepak; Chakrabarti, Saikat; Chattopadhyay, Sharmila

2016-09-01

Podophyllotoxin (ptox) is a therapeutically important lignan derived from Podophyllum hexandrum and is used as a precursor for the synthesis of anticancer drugs etoposide, teniposide and etopophose. In spite of its enormous economic significance, genomic information on this endangered medicinal herb is scarce. We have performed de novo transcriptome analysis of methyl jasmonate (MeJA)-treated P. hexandrum cell cultures exhibiting enhanced ptox accumulation. The results revealed the maximum up-regulation of several isoforms of cinnamyl alcohol dehydrogenase (CAD). CAD catalyzes the synthesis of coniferyl alcohol and sinapyl alcohol from coniferaldehyde (CAld) and sinapaldehyde respectively. Coniferyl alcohol can produce both lignin and lignan while sinapyl alcohol produces only lignin. To isolate the CAD isoforms favoring ptox, we deduced full length cDNA sequences of four CAD isoforms: PhCAD1, PhCAD2, PhCAD3 and PhCAD4 from the contigs of the transcriptome data. In vitro enzyme assays indicated a higher affinity for CAld over sinapaldehyde for each isoform. In silico molecular docking analyses also suggested that PhCAD3 has a higher binding preference with CAld over sinapaldehyde, followed by PhCAD4, PhCAD2, and PhCAD1, respectively. The transgenic cell cultures overexpressing these isoforms independently revealed that PhCAD3 favored the maximum accumulation of ptox as compared to lignin followed by PhCAD4 and PhCAD2, whereas, PhCAD1 favored both equally. Together, our study reveals transcriptome-wide identification and characterization of ptox specific CAD isoforms from P. hexandrum. It provides a useful resource for future research not only on the ptox biosynthetic pathway but on overall P. hexandrum, an endangered medicinal herb with immense therapeutic importance.

The predominant WT1 isoform (+KTS) encodes a DNA-binding protein targeting the planar cell polarity gene Scribble in renal podocytes.

Science.gov (United States)

Wells, Julie; Rivera, Miguel N; Kim, Woo Jae; Starbuck, Kristen; Haber, Daniel A

2010-07-01

WT1 encodes a tumor suppressor first identified by its inactivation in Wilms' Tumor. Although one WT1 splicing variant encodes a well-characterized zinc finger transcription factor, little is known about the function of the most prevalent WT1 isoform, whose DNA binding domain is disrupted by a three-amino acid (KTS) insertion. Using cells that conditionally express WT1(+KTS), we undertook a genome-wide chromatin immunoprecipitation and cloning analysis to identify candidate WT1(+KTS)-regulated promoters. We identified the planar cell polarity gene Scribble (SCRB) as the first WT1(+KTS) target gene in podocytes of the kidney. WT1 and SCRB expression patterns overlap precisely in developing renal glomeruli of mice, and WT1(+KTS) binds to a 33-nucleotide region within the Scribble promoter in mouse and human cell lines and kidneys. Together, our results support a role for the predominant WT1(+KTS) isoform in transcriptional regulation and suggest a link between the WT1-dependent tumor suppressor pathway and a key component of the planar cell polarity pathway.

The predominant WT1 isoform (+KTS) encodes a DNA binding protein targeting the planar cell polarity gene Scribble in renal podocytes

Science.gov (United States)

Wells, Julie; Rivera, Miguel N.; Kim, Woo Jae; Starbuck, Kristen; Haber, Daniel A.

2010-01-01

WT1 encodes a tumor suppressor, first identified by its inactivation in Wilms Tumor. While one WT1 splicing variant encodes a well-characterized zinc finger transcription factor, little is known about the function of the most prevalent WT1 isoform, whose DNA binding domain is disrupted by a three amino acid (KTS) insertion. Using cells which conditionally express WT1(+KTS), we undertook a genome-wide chromatin immunoprecipitation and cloning (ChIP-cloning) analysis to identify candidate WT1(+KTS) regulated promoters. We identified the planar cell polarity (PCP) gene Scribble (SCRB) as the first WT1(+KTS) target gene in podocytes of the kidney. WT1 and SCRB expression patterns overlap precisely in developing renal glomeruli of mice, and WT1(+KTS) binds to a 33 nucleotide region within the Scribble promoter in both mouse and human cell lines and kidneys. Together, our results support a role for the predominant WT1(+KTS) isoform in transcriptional regulation and suggest a link between the WT1-dependent tumor suppressor pathway and a key component of the planar cell polarity pathway. PMID:20571064

Cooperation between two ClpB isoforms enhances the recovery of the recombinant {beta}-galactosidase from inclusion bodies

Energy Technology Data Exchange (ETDEWEB)

Guenther, Izabela [Department of Biochemistry, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk (Poland); Zolkiewski, Michal [Department of Biochemistry, Kansas State University, Manhattan, KS 66506 (United States); Kedzierska-Mieszkowska, Sabina, E-mail: kedzie@biotech.ug.gda.pl [Department of Biochemistry, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk (Poland)

2012-10-05

Highlights: Black-Right-Pointing-Pointer An important role of synergistic cooperation between the two ClpB isoforms. Black-Right-Pointing-Pointer Both ClpB isoforms are associated with IBs of {beta}-galactosidase. Black-Right-Pointing-Pointer ClpB is a key chaperone in IB protein release. -- Abstract: Bacterial ClpB is a molecular chaperone that solubilizes and reactivates aggregated proteins in cooperation with the DnaK chaperone system. The mechanism of protein disaggregation mediated by ClpB is linked to translocation of substrates through the central channel within the ring-hexameric structure of ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and the truncated ClpB80 (ClpB{Delta}N), which does not contain the N-terminal domain. The functional specificity of the two ClpB isoforms and the biological role of the N-terminal domain are still not fully understood. Recently, it has been demonstrated that ClpB may achieve its full potential as an aggregate-reactivating chaperone through the functional interaction and synergistic cooperation of its two isoforms. It has been found that the most efficient resolubilization and reactivation of stress-aggregated proteins occurred in the presence of both ClpB95 and ClpB80. In this work, we asked if the two ClpB isoforms functionally cooperate in the solubilization and reactivation of proteins from insoluble inclusion bodies (IBs) in Escherichia coli cells. Using the model {beta}-galactosidase fusion protein (VP1LAC), we found that solubilization and reactivation of enzymes entrapped in IBs occurred more efficiently in the presence of ClpB95 with ClpB80 than with either ClpB95 or ClpB80 alone. The two isoforms of ClpB chaperone acting together enhanced the solubility and enzymatic activity of {beta}-galactosidase sequestered into IBs. Both ClpB isoforms were associated with IBs of {beta}-galactosidase, what demonstrates their affinity to this type of aggregates. These results demonstrate a synergistic

Differential expression of mRNAs for protein kinase inhibitor isoforms in mouse brain.

OpenAIRE

Seasholtz, A F; Gamm, D M; Ballestero, R P; Scarpetta, M A; Uhler, M D

1995-01-01

Many neurotransmitters are known to regulate neuronal cell function by means of activation of cAMP-dependent protein kinase (PKA) and phosphorylation of neuronal substrate proteins, including transcription factors and ion channels. Here, we have characterized the gene expression of two isoforms of a protein kinase inhibitor (PKI) specific for PKA in mouse brain by RNase protection and in situ hybridization histochemistry. The studies demonstrate that the PKI alpha isoform is abundant in many ...

Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis.

Science.gov (United States)

Kawai, Yoshikazu; Asai, Kei; Errington, Jeffery

2009-08-01

MreB proteins are bacterial actin homologues thought to have a role in cell shape determination by positioning the cell wall synthetic machinery. Many bacteria, particularly Gram-positives, have more than one MreB isoform. Bacillus subtilis has three, MreB, Mbl and MreBH, which colocalize in a single helical structure. We now show that the helical pattern of peptidoglycan (PG) synthesis in the cylindrical part of the rod-shaped cell is governed by the redundant action of the three MreB isoforms. Single mutants for any one of mreB isoforms can still incorporate PG in a helical pattern and generate a rod shape. However, after depletion of MreB in an mbl mutant (or depletion of all three isoforms) lateral wall PG synthesis was impaired and the cells became spherical and lytic. Overexpression of any one of the MreB isoforms overcame the lethality as well as the defects in lateral PG synthesis and cell shape. Furthermore, MreB and Mbl can associate with the peptidoglycan biosynthetic machinery independently. However, no single MreB isoform was able to support normal growth under various stress conditions, suggesting that the multiple isoforms are used to allow cells to maintain proper growth and morphogenesis under changing and sometimes adverse conditions.

Growth hormone receptor exon 3 isoforms may have no importance in the clinical setting of multiethnic Brazilian acromegaly patients.

Science.gov (United States)

de Oliveira Machado, Evelyn; Lima, Carlos Henrique Azeredo; Ogino, Liana Lumi; Kasuki, Leandro; Gadelha, Mônica R

2016-08-01

Acromegaly is associated with significant morbidity and increased mortality, but has a variable severity phenotype. The presence of the exon 3-deleted isoform of the growth hormone receptor (d3-GHR) may influence the disease phenotype and treatment outcomes, including the frequency of biochemical discordance after medical treatment. The objective of this study was to analyze the influence of the d3-GHR isoform on clinical and biochemical characteristics and in the treatment outcomes of Brazilian multiethnic acromegaly patients. We retrospectively analyzed our acromegaly outpatient clinic databank and collected demographic, clinical, biochemical and treatment outcome data from those patients who agreed to participate in the study. A blood sample was collected from all patients, the DNA was extracted and the GHR isoforms were evaluated by PCR, with the full length (fl)-GHR represented by a 935-bp fragment and the d3-GHR represented by a 532-bp fragment. A total of 121 patients were included. Fifty-six patients (46.3 %) were full-length homozygous (fl/fl), 48 (39.7 %) were heterozygous (fl/d3) and 17 (14.0 %) were d3-GHR homozygous (d3/d3). There was no difference between patients homozygous for the fl isoform and those harboring at least one d3-GHR allele in the demographic, clinical and biochemical data or in the treatment outcomes, including somatostatin receptor ligands (SRL) monotherapy, combination therapy with SRL and cabergoline and pegvisomant treatment. There was also no difference between the groups for the frequency of GH and IGF-I discordance after medical treatment. GHR exon 3 genotyping appears to have no clinical significance, at least in Brazilian acromegaly patients.

Roles of the troponin isoforms during indirect flight muscle ...

Indian Academy of Sciences (India)

IFMs) undergo post-transcriptional and post-translational isoform changes during pupal to adult metamorphosis to meet the high energy and mechanical demands of flight. Using a newly generated Gal4 strain (UH3-Gal4) which is expressed ...

A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

Science.gov (United States)

Kumar, Priyadarsini; Walsh, Donal A

2002-03-15

We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

The related transcriptional enhancer factor-1 isoform, TEAD4(216, can repress vascular endothelial growth factor expression in mammalian cells.

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Binoy Appukuttan

Full Text Available Increased cellular production of vascular endothelial growth factor (VEGF is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4 protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4(216, which represses VEGF promoter activity. The TEAD4(216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE, which is the sequence critical to hypoxia inducible factor (HIF-mediated effects. The TEAD4(216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4(216 isoform can competitively repress the stimulatory activity of the TEAD4(434 and TEAD4(148 enhancers. Synthesis of the native VEGF(165 protein and cellular proliferation is suppressed by the TEAD4(216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4(216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases.

Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing.

Science.gov (United States)

Huertas, César S; Carrascosa, L G; Bonnal, S; Valcárcel, J; Lechuga, L M

2016-04-15

Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

The effect of a period of intensive exercise on the isoform test to detect growth hormone doping in sports.

Science.gov (United States)

Voss, S C; Giraud, S; Alsayrafi, M; Bourdon, P C; Schumacher, Y O; Saugy, M; Robinson, N

2013-08-01

The major objective of this study was to investigate the effects of several days of intense exercise on growth hormone (hGH) testing using the World Anti-Doping Agencies hGH isoform differential immunoassays. Additionally the effects of circadian variation and exercise type on the isoform ratios were also investigated. 15 male athletes performed a simulated nine day cycling stage race. Blood samples were collected twice daily over a period of 15 days (stage race+three days before and after). hGH isoforms were analysed by the official WADA immunoassays (CMZ Assay GmbH). All measured isoform ratios were far below the WADA decision limits for an adverse analytical finding. Changes in the isoform ratios could not be clearly connected to circadian variation, exercise duration or intensity. The present study demonstrates that the hGH isoform ratios are not significantly affected by exercise or circadian variation. We demonstrated that heavy, long term exercise does not interfere with the decision limits for an adverse analytical finding. Copyright © 2013 Elsevier Ltd. All rights reserved.

Splice form variant and amino acid changes in MDR49 confers DDT resistance in transgenic Drosophila.

Science.gov (United States)

Seong, Keon Mook; Sun, Weilin; Clark, John M; Pittendrigh, Barry R

2016-03-22

The ATP-binding cassette (ABC) transporters represent a superfamily of proteins that have important physiological roles in both prokaryotes and eukaryotes. In insects, ABC transporters have previously been implicated in insecticide resistance. The 91-R strain of Drosophila melanogaster has been intensely selected with DDT over six decades. A recent selective sweeps analysis of 91-R implicated the potential role of MDR49, an ABC transporter, in DDT resistance, however, to date the details of how MDR49 may play a role in resistance have not been elucidated. In this study, we investigated the impact of structural changes and an alternative splicing event in MDR49 on DDT-resistance in 91-R, as compared to the DDT susceptible strain 91-C. We observed three amino acid differences in MDR49 when 91-R was compared with 91-C, and only one isoform (MDR49B) was implicated in DDT resistance. A transgenic Drosophila strain containing the 91-R-MDR49B isoform had a significantly higher LD50 value as compared to the 91-C-MDR49B isoform at the early time points (6 h to 12 h) during DDT exposure. Our data support the hypothesis that the MDR49B isoform, with three amino acid mutations, plays a role in the early aspects of DDT resistance in 91-R.

Isoform-specific potentiation of stem and progenitor cell engraftment by AML1/RUNX1.

Directory of Open Access Journals (Sweden)

Shinobu Tsuzuki

2007-05-01

Full Text Available AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo.The human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo.These data demonstrate that the "a" isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of leukaemia and also in cord blood

Characterization of the expression of the pro-metastatic Mena(INV) isoform during breast tumor progression.

Science.gov (United States)

Oudin, Madeleine J; Hughes, Shannon K; Rohani, Nazanin; Moufarrej, Mira N; Jones, Joan G; Condeelis, John S; Lauffenburger, Douglas A; Gertler, Frank B

2016-03-01

Several functionally distinct isoforms of the actin regulatory Mena are produced by alternative splicing during tumor progression. Forced expression of the Mena(INV) isoform drives invasion, intravasation and metastasis. However, the abundance and distribution of endogenously expressed Mena(INV) within primary tumors during progression remain unknown, as most studies to date have only assessed relative mRNA levels from dissociated tumor samples. We have developed a Mena(INV) isoform-specific monoclonal antibody and used it to examine Mena(INV) expression patterns in mouse mammary and human breast tumors. Mena(INV) expression increases during tumor progression and to examine the relationship between Mena(INV) expression and markers for epithelial or mesenchymal status, stemness, stromal cell types and hypoxic regions. Further, while Mena(INV) robustly expressed in vascularized areas of the tumor, it is not confined to cells adjacent to blood vessels. Altogether, these data demonstrate the specificity and utility of the anti-Mena(INV)-isoform specific antibody, and provide the first description of endogenous Mena(INV) protein expression in mouse and human tumors.

Modified Polyadenylation-Based RT-qPCR Increases Selectivity of Amplification of 3′-MicroRNA Isoforms

Directory of Open Access Journals (Sweden)

Charlotte Nejad

2018-01-01

Full Text Available MicroRNA (miRNA detection by reverse transcription (RT quantitative real-time PCR (RT-qPCR is the most popular method currently used to measure miRNA expression. Although the majority of miRNA families are constituted of several 3′-end length variants (“isomiRs”, little attention has been paid to their differential detection by RT-qPCR. However, recent evidence indicates that 3′-end miRNA isoforms can exhibit 3′-length specific regulatory functions, underlining the need to develop strategies to differentiate 3′-isomiRs by RT-qPCR approaches. We demonstrate here that polyadenylation-based RT-qPCR strategies targeted to 20–21 nt isoforms amplify entire miRNA families, but that primers targeted to >22 nt isoforms were specific to >21 nt isoforms. Based on this observation, we developed a simple method to increase selectivity of polyadenylation-based RT-qPCR assays toward shorter isoforms, and demonstrate its capacity to help distinguish short RNAs from longer ones, using synthetic RNAs and biological samples with altered isomiR stoichiometry. Our approach can be adapted to many polyadenylation-based RT-qPCR technologies already exiting, providing a convenient way to distinguish long and short 3′-isomiRs.

DMPD: The role of C/EBP isoforms in the control of inflammatory and native immunityfunctions. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 9792624 The role of C/EBP isoforms in the control of inflammatory and native immunityfunction...f C/EBP isoforms in the control of inflammatory and native immunityfunctions. PubmedID 9792624 Title The rol...e of C/EBP isoforms in the control of inflammatory and native immunityfunctions.

Enantioselective radical reactions. Evaluation of nitrogen protecting groups in the synthesis of beta-amino acids.

Science.gov (United States)

Sibi, Mukund P; Patil, Kalyani

2006-02-20

We have investigated the effect of nitrogen protecting groups in radical addition trapping experiments leading to beta(2)-amino acids. Of the three N-protecting groups examined, the phthalimido group was optimal with respect to both yields and enantioselectivity. Additionally, radical additions to more complex acrylates were also investigated, which provided access to functionalized beta(2)-amino acids in modest selectivity.

Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

Science.gov (United States)

Hector, Ralph D; Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

2016-01-01

Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

Echinococcus granulosus fatty acid binding proteins subcellular localization.

Science.gov (United States)

Alvite, Gabriela; Esteves, Adriana

2016-05-01

Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

Ginger extract mitigates ethanol-induced changes of alpha and beta - myosin heavy chain isoforms gene expression and oxidative stress in the heart of male wistar rats.

Science.gov (United States)

Shirpoor, Alireza; Zerehpoosh, Mitra; Ansari, Mohammad Hasan Khadem; Kheradmand, Fatemeh; Rasmi, Yousef

2017-09-01

The association between ethanol consumption and heart abnormalities, such as chamber dilation, myocyte damage, ventricular hypertrophy, and hypertension is well known. However, underlying molecular mediators involved in ethanol-induced heart abnormalities remain elusive. The aim of this study was to investigate the effect of chronic ethanol exposure on alpha and beta - myosin heavy chain (MHC) isoforms gene expression transition and oxidative stress in rats' heart. It was also planned to find out whether ginger extract mitigated the abnormalities induced by ethanol in rats' heart. Male wistar rats were divided into three groups of eight animals as follows: Control, ethanol, and ginger extract treated ethanolic (GETE) groups. After six weeks of treatment, the results revealed a significant increase in the β-MHC gene expression, 8- OHdG amount, and NADPH oxidase level. Furthermore, a significant decrease in the ratio of α-MHC/β-MHC gene expression to the amount of paraoxonase enzyme in the ethanol group compared to the control group was found. The consumption of Ginger extract along with ethanol ameliorated the changes in MHC isoforms gene expression and reduced the elevated amount of 8-OHdG and NADPH oxidase. Moreover, compared to the consumption of ethanol alone, it increased the paraoxonase level significantly. These findings indicate that ethanol-induced heart abnormalities may in part be associated with MHC isoforms changes mediated by oxidative stress, and that these effects can be alleviated by using ginger extract as an antioxidant molecule. Copyright © 2017 Elsevier B.V. All rights reserved.

Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents

Science.gov (United States)

Benoit, Stephen C.; Kemp, Christopher J.; Elias, Carol F.; Abplanalp, William; Herman, James P.; Migrenne, Stephanie; Lefevre, Anne-Laure; Cruciani-Guglielmacci, Céline; Magnan, Christophe; Yu, Fang; Niswender, Kevin; Irani, Boman G.; Holland, William L.; Clegg, Deborah J.

2009-01-01

Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-θ was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-θ to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-θ nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-θ attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-θ activation, resulting in reduced insulin activity. PMID:19726875

Palmitic acid mediates hypothalamic insulin resistance by altering PKC-theta subcellular localization in rodents.

Science.gov (United States)

Benoit, Stephen C; Kemp, Christopher J; Elias, Carol F; Abplanalp, William; Herman, James P; Migrenne, Stephanie; Lefevre, Anne-Laure; Cruciani-Guglielmacci, Céline; Magnan, Christophe; Yu, Fang; Niswender, Kevin; Irani, Boman G; Holland, William L; Clegg, Deborah J

2009-09-01

Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-theta, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-theta was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-theta to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-theta nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-theta attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-theta activation, resulting in reduced insulin activity.

Efficacy of Highly Purified Urinary FSH versus Recombinant FSH in Chinese Women over 37 Years Undergoing Assisted Reproductive Techniques

Directory of Open Access Journals (Sweden)

Xuemei Liu

2015-02-01

Full Text Available Background: Urine derived follicle-stimulating hormone (uFSH contains a higher proportion of acidic isoforms, whereas recombinant FSH (rFSH contains a higher proportion of less-acidic isoforms. Less-acidic isoforms have a faster clearance, and thus a shorter half-life than the acidic FSH isoforms. The slow clearance of the acidic isoforms has a longer half-life and higher biological activity. This study was designed to determine whether uFSH or rFSH is more effective in older Chinese women undergoing assisted reproductive techniques (ART. Materials and Methods: This is a prospective, randomized, controlled cohort study. A total of 508 Chinese women over 37 years were randomized into two following study groups for their in vitro fertilization (IVF or intracytoplasmic sperm injection (ICSI cycles: i. group A (n=254 were treated with rFSH, and ii. group B (n=254 were treated with uFSH. Both groups were suppressed with a gonadotropin-releasing hormone (GnRH analogue using a long down-regulation protocol. The main outcomes for comparison were days of stimulation, estradiol (E2 on the day of human chorionic gonadotropin (hCG administration, number of oocytes collected, amount of FSH used, quantity of FSH/oocyte, endometrial thickness at hCG day, M П oocyte rate, 2PN zygote rate, grade І embryo rate, number of embryos cryopreserved, pregnancy rate, implantation rate, abortion rate and the rate of no transferable embryos. Results: Twenty two cycles including 16 cycles with poor ovarian response and six cycles with ovarian hyperstimulation syndrome were cancelled. There were 243 cycles left in each group. The patients treated with uFSH had a significantly higher 2PN zygote rate (87.4 vs. 76.6%, p0.05. Conclusion: This study showed that uFSH produced a significantly higher proportion of grade І embryos than rFSH in older Chinese women and there was a significantly lower chance of no transferable embryos in uFSH cycles. The clinical efficacy of the two

The three isoforms of the light-harvesting complex II Spectroscopic features, trimer formation, and functional roles

CERN Document Server

Standfuss, Jorg

2004-01-01

The major light-harvesting complex (LHC-II) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus. Native LHC-II isolated from plant tissue consists of three isoforms, Lhcb1, Lhcb2, and Lhcb3, which form homo- and heterotrimers. All three isoforms are highly conserved among different species, suggesting distinct functional roles. We produced the three LHC-II isoforms by heterologous expression of the polypeptide in Escherichia coli and in vitro refolding with purified pigments. Although Lhcb1 and Lhcb2 are very similar in polypeptide sequence and pigment content, Lhcb3 is clearly different because it lacks an N-terminal phosphorylation site and has a higher chlorophyll a/b ratio, suggesting the absence of one chlorophyll b. Low temperature absorption and fluorescence emission spectra of the pure isoforms revealed small but significant differences in pigment organization. The oligomeric state of the pure isofo...

CD44 isoforms are heterogeneously expressed in breast cancer and correlate with tumor subtypes and cancer stem cell markers

International Nuclear Information System (INIS)

Olsson, Eleonor; Lövgren, Kristina; Fernö, Mårten; Grabau, Dorthe; Borg, Åke; Hegardt, Cecilia; Honeth, Gabriella; Bendahl, Pär-Ola; Saal, Lao H; Gruvberger-Saal, Sofia; Ringnér, Markus; Vallon-Christersson, Johan; Jönsson, Göran; Holm, Karolina

2011-01-01

The CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and has been implicated in the metastatic process as well as in the putative cancer stem cell (CSC) compartment. We aimed to investigate potential associations between alternatively spliced isoforms of CD44 and CSCs as well as to various breast cancer biomarkers and molecular subtypes. We used q-RT-PCR and exon-exon spanning assays to analyze the expression of four alternatively spliced CD44 isoforms as well as the total expression of CD44 in 187 breast tumors and 13 cell lines. ALDH1 protein expression was determined by IHC on TMA. Breast cancer cell lines showed a heterogeneous expression pattern of the CD44 isoforms, which shifted considerably when cells were grown as mammospheres. Tumors characterized as positive for the CD44 + /CD24 - phenotype by immunohistochemistry were associated to all isoforms except the CD44 standard (CD44S) isoform, which lacks all variant exons. Conversely, tumors with strong expression of the CSC marker ALDH1 had elevated expression of CD44S. A high expression of the CD44v2-v10 isoform, which retain all variant exons, was correlated to positive steroid receptor status, low proliferation and luminal A subtype. The CD44v3-v10 isoform showed similar correlations, while high expression of CD44v8-v10 was correlated to positive EGFR, negative/low HER2 status and basal-like subtype. High expression of CD44S was associated with strong HER2 staining and also a subgroup of basal-like tumors. Unsupervised hierarchical cluster analysis of CD44 isoform expression data divided tumors into four main clusters, which showed significant correlations to molecular subtypes and differences in 10-year overall survival. We demonstrate that individual CD44 isoforms can be associated to different breast cancer subtypes and clinical markers such as HER2, ER and PgR, which suggests involvement of CD44 splice variants in specific oncogenic signaling pathways. Efforts to link CD44 to

CD44 isoforms are heterogeneously expressed in breast cancer and correlate with tumor subtypes and cancer stem cell markers

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Vallon-Christersson Johan

2011-09-01

Full Text Available Abstract Background The CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and has been implicated in the metastatic process as well as in the putative cancer stem cell (CSC compartment. We aimed to investigate potential associations between alternatively spliced isoforms of CD44 and CSCs as well as to various breast cancer biomarkers and molecular subtypes. Methods We used q-RT-PCR and exon-exon spanning assays to analyze the expression of four alternatively spliced CD44 isoforms as well as the total expression of CD44 in 187 breast tumors and 13 cell lines. ALDH1 protein expression was determined by IHC on TMA. Results Breast cancer cell lines showed a heterogeneous expression pattern of the CD44 isoforms, which shifted considerably when cells were grown as mammospheres. Tumors characterized as positive for the CD44+/CD24- phenotype by immunohistochemistry were associated to all isoforms except the CD44 standard (CD44S isoform, which lacks all variant exons. Conversely, tumors with strong expression of the CSC marker ALDH1 had elevated expression of CD44S. A high expression of the CD44v2-v10 isoform, which retain all variant exons, was correlated to positive steroid receptor status, low proliferation and luminal A subtype. The CD44v3-v10 isoform showed similar correlations, while high expression of CD44v8-v10 was correlated to positive EGFR, negative/low HER2 status and basal-like subtype. High expression of CD44S was associated with strong HER2 staining and also a subgroup of basal-like tumors. Unsupervised hierarchical cluster analysis of CD44 isoform expression data divided tumors into four main clusters, which showed significant correlations to molecular subtypes and differences in 10-year overall survival. Conclusions We demonstrate that individual CD44 isoforms can be associated to different breast cancer subtypes and clinical markers such as HER2, ER and PgR, which suggests involvement of CD44 splice variants in

Wnt isoform-specific interactions with coreceptor specify inhibition or potentiation of signaling by LRP6 antibodies.

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Yan Gong

Full Text Available β-Catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may

Quantitative determination of acidic groups in functionalized graphene by direct titration

Czech Academy of Sciences Publication Activity Database

Ederer, J.; Janoš, P.; Ecorchard, Petra; Štengl, Václav; Bělčická, Zuzana; Šťastný, Martin; Pop-Georgievski, Ognen; Dohnal, V.

2016-01-01

Roč. 103, JUN (2016), s. 44-53 ISSN 1381-5148 R&D Projects: GA ČR(CZ) GA14-05146S Institutional support: RVO:61388980 ; RVO:61389013 Keywords : Acid-base titration * Carboxylic functional groups * Graphene oxide * Phenolic functional groups * Proton-binding model Subject RIV: CA - Inorganic Chemistry ; CD - Macromolecular Chemistry (UMCH-V) Impact factor: 3.151, year: 2016

Effect of 48-h food deprivation on the expressions of myosin heavy-chain isoforms and fiber type-related factors in rats.

Science.gov (United States)

Mizunoya, Wataru; Sawano, Shoko; Iwamoto, Yohei; Sato, Yusuke; Tatsumi, Ryuichi; Ikeuchi, Yoshihide

2013-01-01

The primary aim of this study was to examine the effects of 48-h food deprivation on rat skeletal muscle fiber type, according to myosin heavy-chain (MyHC) isoform composition and some metabolism-related factors in both slow-type dominant and fast-type dominant muscle tissues. Male Wistar rats (7 wk old) were treated with 48-h food deprivation or ad libitum feeding as control. After the treatment, the soleus muscle (slow-type dominant) and the extensor digitorum longus (EDL, fast-type dominant) were excised. We found that 48-h food deprivation did not affect MyHC composition in either the soleus or EDL, compared with fed rats by electrophoretic separation of MyHC isoforms. However, 48-h food deprivation significantly increased the mRNA expression of fast-type MyHC2B in the EDL muscle. Moreover, food deprivation increased fatty acid metabolism, as shown by elevated levels of related serum energy substrates and mRNA expression of mitochondrial uncoupling protein (UCP) 3 and lipoprotein lipase (LPL) in both the soleus and EDL. UCP3 and LPL are generally expressed at higher levels in slow-type fibers. Furthermore, we found that food deprivation significantly decreased the protein amounts of PGC1α and phosphorylated FOXO1, which are known as skeletal muscle fiber type regulators. In conclusion, 48-h food deprivation increased mRNA expression of fast-type MyHC isoform and oxidative metabolism-related factors in EDL, whereas MyHC composition at the protein level did not change in either the soleus or EDL.

Acidic pH and short-chain fatty acids activate Na+ transport but differentially modulate expression of Na+/H+ exchanger isoforms 1, 2, and 3 in omasal epithelium.

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Lu, Zhongyan; Yao, Lei; Jiang, Zhengqian; Aschenbach, Jörg R; Martens, Holger; Shen, Zanming

2016-01-01

Low sodium content in feed and large amounts of salivary sodium secretion are essential requirements to efficient sodium reabsorption in the dairy cow. It is already known that Na(+)/H(+) exchange (NHE) of the ruminal epithelium plays a key role in Na(+) absorption, and its function is influenced by the presence of short-chain fatty acids (SCFA) and mucosal pH. By contrast, the functional role and regulation of NHE in omasal epithelium have not been completely understood. In the present study, we used model studies in small ruminants (sheep and goats) to investigate NHE-mediated Na(+) transport and the effects of pH and SCFA on NHE activity in omasal epithelium and on the expression of NHE isoform in omasal epithelial cells. Conventional Ussing chamber technique, primary cell culture, quantitative PCR, and Western blot were used. In native omasal epithelium of sheep, the Na(+) transport was electroneutral, and it was inhibited by the specific NHE3 inhibitor 3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride, which decreased mucosal-to-serosal, serosal-to-mucosal, and net flux rates of Na(+) by 80% each. The application of low mucosal pH (6.4 or 5.8) in the presence of SCFA activated the Na(+) transport across omasal epithelium of sheep compared with that at pH 7.4. In cultured omasal epithelial cells of goats, mRNA and protein of NHE1, NHE2, and NHE3 were detected. The application of SCFA increased NHE1 mRNA and protein expression, which was most prominent when the culture medium pH decreased from 7.4 to 6.8. At variance, the mRNA and protein expression of NHE2 and NHE3 were decreased with low pH and SCFA, which was contrary to the published data from ruminal epithelial studies. In conclusion, this paper shows that (1) NHE1, NHE2, and NHE3 are expressed in omasal epithelium; (2) NHE3 mediates the major portion of transepithelial Na(+) transport in omasal epithelium; and (3) SCFA and acidic pH acutely

A route for oxygen isotope enrichment of α-COOH groups in amino acids

International Nuclear Information System (INIS)

Steinschneidner, A.; St Armour, T.; Valentine, B.; Burgar, M.I.; Fiat, D.

1981-01-01

Oxygen-17 was introduced into leucine, proline, phenylalanine and tyrosine. The corresponding tert-butyloxycarbonyl amino acids were first converted to their O-methyl esters. Following saponification with Na 17 OH, the tert-butyloxycarbonyl group was removed to yield free amino acid enriched with oxygen-17 by approximately 1000-fold. Oxygen-17 NMR revealed well-resolved peaks for the labelled amino acids. The chemical shifts are reported. (author)

Molecular characterization of two isoforms of ZFAND3 cDNA from the Japanese quail and the leopard gecko, and different expression patterns between testis and ovary.

Science.gov (United States)

Otake, Shigeo; Endo, Daisuke; Park, Min Kyun

2011-11-15

Zing finger AN1-type domain 3 (ZFAND3), also known as testis expressed sequence 27 (Tex27), is a gene found in the mouse testis, but its physiological function is unknown. We identified the full-length sequences of two isoforms (short and long) of ZFAND3 cDNA from Japanese quail and leopard gecko. This is the first cloning of avian and reptilian ZFAND3 cDNA. The two isoforms are generated by alternative polyadenylation in the 3'UTR and have the same ORF sequences encoding identical proteins. There were highly conserved regions in the 3'UTR of the long form near the polyadenylation sites from mammals to amphibians, suggesting that the features for determining the stability of mRNA or translation efficiency differ between isoforms. The deduced amino acid sequence of ZFAND3 has two putative zinc finger domains, an A20-like zinc finger domain at the N-terminal and an AN1-like zinc finger domain at the C-terminal. Sequence analysis revealed an additional exon in the genomic structures of the avian and reptilian ZFAND3 genes which is not present in mammals, amphibians, or fish, and this exon produces additional amino acid residues in the A20-like zinc finger domain. Expression analysis in Japanese quail revealed that the expression level of ZFAND3 mRNA was high in not only the testis but also the ovary, and ZFAND3 mRNA was expressed in both spermatides of the testis and oocytes of the ovary. While the short form mRNA was mainly expressed in the testis, the expression level of the long form mRNA was high in the ovary. These results suggest that ZFAND3 has physiological functions related to germ cell maturation and regulatory mechanisms that differ between the testis and ovary. Copyright © 2011 Elsevier B.V. All rights reserved.

Metabolomic Discovery of Novel Urinary Galabiosylceramide Analogs as Fabry Disease Biomarkers

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Boutin, Michel; Auray-Blais, Christiane

2015-03-01

Fabry disease is an X-linked, complex, multisystemic lysosomal storage disorder presenting marked phenotypic and genotypic variability among affected male and female patients. Glycosphingolipids, mainly globotriaosylceramide (Gb3) isoforms/analogs, globotriaosylsphingosine (lyso-Gb3) and analogs, as well as galabiosylceramide (Ga2) isoforms/analogs accumulate in the vascular endothelium, nerves, cardiomyocytes, renal glomerular and tubular epithelial cells, and biological fluids. The search for biomarkers reflecting disease severity and progression is still on-going. A metabolomic study using quadrupole time-of-flight mass spectrometry has revealed 22 galabiosylceramide isoforms/analogs in urine of untreated Fabry patients classified in seven groups according to their chemical structure: (1) Saturated fatty acid; (2) one extra double bond; (3) two extra double bonds; (4) hydroxylated saturated fatty acid; (5) hydroxylated fatty acid and one extra double bond; (6) hydrated sphingosine and hydroxylated fatty acid; (7) methylated amide linkage. Relative quantification of both Ga2 and Gb3 isoforms/analogs was performed. All these biomarkers are significantly more abundant in urine samples from untreated Fabry males compared with healthy male controls. A significant amount of Ga2 isoforms/analogs, accounting for 18% of all glycosphingolipids analyzed (Ga2 + Gb3 and respective isoforms/analogs), were present in urine of Fabry patients. Gb3 isoforms containing saturated fatty acids are the most abundant (60.9%) compared with 26.3% for Ga2. A comparison between Ga2 isoforms/analogs and their Gb3 counterparts also showed that the proportion of analogs with hydroxylated fatty acids is significantly greater for Ga2 (35.8%) compared with Gb3 (1.9%). These results suggest different biological pathways involved in the synthesis and/or degradation of Gb3 and Ga2 metabolites.

Photoacidic and Photobasic Behavior of Transition Metal Compounds with Carboxylic Acid Group(s)

Energy Technology Data Exchange (ETDEWEB)

O’Donnell, Ryan M. [Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States; Sampaio, Renato N. [Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States; Li, Guocan [Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States; Johansson, Patrik G. [Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States; Ward, Cassandra L. [Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States; Meyer, Gerald J. [Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States

2016-03-10

Excited state proton transfer studies of six Ru polypyridyl compounds with carboxylic acid/carboxylate group(s) revealed that some were photoacids and some were photobases. The compounds [Ru^II(btfmb)₂(LL)]²⁺, [Ru^II(dtb)2(LL)]2+, and [RuII(bpy)2(LL)]2+, where bpy is 2,2'-bipyridine, btfmb is 4,4'-(CF₃)₂-bpy, and dtb is 4,4'-((CH₃)₃C)₂-bpy, and LL is either dcb = 4,4'-(CO₂H)₂-bpy or mcb = 4-(CO₂H),4'-(CO2Et)-2,2'-bpy, were synthesized and characterized. The compounds exhibited intense metal-to-ligand charge-transfer (MLCT) absorption bands in the visible region and room temperature photoluminescence (PL) with long τ > 100 ns excited state lifetimes. The mcb compounds had very similar ground state pKa’s of 2.31 ± 0.07, and their characterization enabled accurate determination of the two pK_a values for the commonly utilized dcb ligand, pK_a1 = 2.1 ± 0.1 and pKa2 = 3.0 ± 0.2. Compounds with the btfmb ligand were photoacidic, and the other compounds were photobasic. Transient absorption spectra indicated that btfmb compounds displayed a [Ru^III(btfmb–)L₂]^2+* localized excited state and a [Ru^III(dcb–)L₂]^2+* formulation for all the other excited states. Time dependent PL spectral shifts provided the first kinetic data for excited state proton transfer in a transition metal compound. PL titrations, thermochemical cycles, and kinetic analysis (for the mcb compounds) provided self-consistent pK_a* values. The ability to make a single ionizable group photobasic or photoacidic through ligand design was unprecedented and was understood based on the orientation of the lowest-lying MLCT excited state dipole relative to the ligand that contained the carboxylic acid group(s).

Differential regulation of protein phosphatase 1 (PP1) isoforms in human heart failure and atrial fibrillation.

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Meyer-Roxlau, Stefanie; Lämmle, Simon; Opitz, Annett; Künzel, Stephan; Joos, Julius P; Neef, Stefan; Sekeres, Karolina; Sossalla, Samuel; Schöndube, Friedrich; Alexiou, Konstantin; Maier, Lars S; Dobrev, Dobromir; Guan, Kaomei; Weber, Silvio; El-Armouche, Ali

2017-07-01

Protein phosphatase 1 (PP1) is a key regulator of important cardiac signaling pathways. Dysregulation of PP1 has been heavily implicated in cardiac dysfunctions. Accordingly, pharmacological targeting of PP1 activity is considered for therapeutic intervention in human cardiomyopathies. Recent evidence from animal models implicated previously unrecognized, isoform-specific activities of PP1 in the healthy and diseased heart. Therefore, this study examined the expression of the distinct PP1 isoforms PP1α, β, and γ in human heart failure (HF) and atrial fibrillation (AF) and addressed the consequences of β-adrenoceptor blocker (beta-blocker) therapy for HF patients with reduced ejection fraction on PP1 isoform expression. Using western blot analysis, we found greater abundance of PP1 isoforms α and γ but unaltered PP1β levels in left ventricular myocardial tissues from HF patients as compared to non-failing controls. However, expression of all three PP1 isoforms was higher in atrial appendages from patients with AF compared to patients with sinus rhythm. Moreover, we found that in human failing ventricles, beta-blocker therapy was associated with lower PP1α abundance and activity, as indicated by higher phosphorylation of the PP1α-specific substrate eIF2α. Greater eIF2α phosphorylation is a known repressor of protein translation, and accordingly, we found lower levels of the endoplasmic reticulum (ER) stress marker Grp78 in the very same samples. We propose that isoform-specific targeting of PP1α activity may be a novel and innovative therapeutic strategy for the treatment of human cardiac diseases by reducing ER stress conditions.

The α and Δ isoforms of CREB1 are required to maintain normal pulmonary vascular resistance.

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Lili Li

Full Text Available Chronic hypoxia causes pulmonary hypertension associated with structural alterations in pulmonary vessels and sustained vasoconstriction. The transcriptional mechanisms responsible for these distinctive changes are unclear. We have previously reported that CREB1 is activated in the lung in response to alveolar hypoxia but not in other organs. To directly investigate the role of α and Δ isoforms of CREB1 in the regulation of pulmonary vascular resistance we examined the responses of mice in which these isoforms of CREB1 had been inactivated by gene mutation, leaving only the β isoform intact (CREB(αΔ mice. Here we report that expression of CREB regulated genes was altered in the lungs of CREB(αΔ mice. CREB(αΔ mice had greater pulmonary vascular resistance than wild types, both basally in normoxia and following exposure to hypoxic conditions for three weeks. There was no difference in rho kinase mediated vasoconstriction between CREB(αΔ and wild type mice. Stereological analysis of pulmonary vascular structure showed characteristic wall thickening and lumen reduction in hypoxic wild-type mice, with similar changes observed in CREB(αΔ. CREB(αΔ mice had larger lungs with reduced epithelial surface density suggesting increased pulmonary compliance. These findings show that α and Δ isoforms of CREB1 regulate homeostatic gene expression in the lung and that normal activity of these isoforms is essential to maintain low pulmonary vascular resistance in both normoxic and hypoxic conditions and to maintain the normal alveolar structure. Interventions that enhance the actions of α and Δ isoforms of CREB1 warrant further investigation in hypoxic lung diseases.

Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia.

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Bhattarai, Sunil; Aly, Ahmed; Garcia, Kristy; Ruiz, Diandra; Pontarelli, Fabrizio; Dharap, Ashutosh

2018-06-03

Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24 h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6 h, 1878 transcripts at 12 h, and 1618 transcripts at 24 h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time-point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprecedented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization.

Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms in the binding of substrates and modulators: probing with chimaeric enzymes.

Science.gov (United States)

Matsuura, K; Hara, A; Deyashiki, Y; Iwasa, H; Kume, T; Ishikura, S; Shiraishi, H; Katagiri, Y

1998-01-01

Human liver dihydrodiol dehydrogenase (DD; EC 1.3.1.20) exists in isoforms (DD1, DD2 and DD4) composed of 323 amino acids. DD1 and DD2 share 98% amino acid sequence identity, but show lower identities (approx. 83%) with DD4, in which a marked difference is seen in the C-terminal ten amino acids. DD4 exhibits unique catalytic properties, such as the ability to oxidize both (R)- and (S)-alicyclic alcohols equally, high dehydrogenase activity for bile acids, potent inhibition by steroidal anti-inflammatory drugs and activation by sulphobromophthalein and clofibric acid derivatives. In this study, we have prepared chimaeric enzymes, in which we exchanged the C-terminal 39 residues between the two enzymes. Compared with DD1, CDD1-4 (DD1 with the C-terminal sequence of DD4) had increased kcat/Km values for 3alpha-hydroxy-5beta-androstanes and bile acids of 3-9-fold and decreased values for the other substrates by 5-100-fold. It also became highly sensitive to DD4 inhibitors such as phenolphthalein and hexoestrol. Another chimaeric enzyme, CDD4-1 (DD4 with the C-terminal sequence of DD1), showed the same (S)-stereospecificity for the alicyclic alcohols as DD1, had decreased kcat/Km values for bile acids with 7beta- or 12alpha-hydroxy groups by more than 120-fold and was resistant to inhibition by betamethasone. In addition, the activation effects of sulphobromophthalein and bezafibrate decreased or disappeared for CDD4-1. The recombinant DD4 with the His314-->Pro (the corresponding residue of DD1) mutation showed intermediate changes in the properties between those of wild-type DD4 and CDD4-1. The results indicate that the binding of substrates, inhibitors and activators to the enzymes is controlled by residues in their C-terminal domains; multiple residues co-ordinately act as determinants for substrate specificity and inhibitor sensitivity. PMID:9820821

Thick filament length and isoform composition determine self-organized contractile units in actomyosin bundles.

Science.gov (United States)

Thoresen, Todd; Lenz, Martin; Gardel, Margaret L

2013-02-05

Diverse myosin II isoforms regulate contractility of actomyosin bundles in disparate physiological processes by variations in both motor mechanochemistry and the extent to which motors are clustered into thick filaments. Although the role of mechanochemistry is well appreciated, the extent to which thick filament length regulates actomyosin contractility is unknown. Here, we study the contractility of minimal actomyosin bundles formed in vitro by mixtures of F-actin and thick filaments of nonmuscle, smooth, and skeletal muscle myosin isoforms with varied length. Diverse myosin II isoforms guide the self-organization of distinct contractile units within in vitro bundles with shortening rates similar to those of in vivo myofibrils and stress fibers. The tendency to form contractile units increases with the thick filament length, resulting in a bundle shortening rate proportional to the length of constituent myosin thick filament. We develop a model that describes our data, providing a framework in which to understand how diverse myosin II isoforms regulate the contractile behaviors of disordered actomyosin bundles found in muscle and nonmuscle cells. These experiments provide insight into physiological processes that use dynamic regulation of thick filament length, such as smooth muscle contraction. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The genetics of feto-placental development: A study of acid phosphatase locus 1 and adenosine deaminase polymorphisms in a consecutive series of newborn infants

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Bergamaschi Antonio

2008-09-01

Full Text Available Abstract Background Acid phosphatase locus 1 and adenosine deaminase locus 1 polymorphisms show cooperative effects on glucose metabolism and immunological functions. The recent observation of cooperation between the two systems on susceptibility to repeated spontaneous miscarriage prompted us to search for possible interactional effects between these genes and the correlation between birth weight and placental weight. Deviation from a balanced development of the feto-placental unit has been found to be associated with perinatal morbidity and mortality and with cardiovascular diseases in adulthood. Methods We examined 400 consecutive newborns from the Caucasian population of Rome. Birth weight, placental weight, and gestational length were registered. Acid phosphatase locus 1 and adenosine deaminase locus 1 phenotypes were determined by starch gel electrophoresis and correlation analysis was performed by SPSS programs. Informed verbal consent to participate in the study was obtained from the mothers. Results Highly significant differences in birth weight-placental weight correlations were observed among acid phosphatase locus 1 phenotypes (p = 0.005. The correlation between birth weight and placental weight was markedly elevated in subjects carrying acid phosphatase locus 1 phenotypes with medium-low F isoform concentration (A, CA and CB phenotypes compared to those carrying acid phosphatase locus 1 phenotypes with medium-high F isoform concentration (BA and B phenotypes (p = 0.002. Environmental and developmental variables were found to exert a significant effect on birth weight-placental weight correlation in subjects with medium-high F isoform concentrations, but only a marginal effect was observed in those with medium-low F isoform concentrations. The correlation between birth weight and placental weight is higher among carriers of the adenosine deaminase locus 1 allele*2, which is associated with low activity, than in homozygous adenosine

Branchial Expression Patterns of Claudin Isoforms in Atlantic Salmon During Seawater Acclimation and Smoltification

DEFF Research Database (Denmark)

Tipsmark, Christian K; Kiilerich, Pia; Nilsen, Tom O

2008-01-01

in epithelia. We identified Atlantic salmon genes belonging to the claudin family by screening expressed sequence tag libraries available at NCBI and classification was performed with aid of maximum likelihood and neighbour-joining analysis. In gill libraries, five isoforms (10e, 27a, 28a, 28b and 30) were...... present and QPCR analysis confirmed tissue-specific expression in gill when compared to kidney, intestine, heart, muscle, brain and liver. Expression patterns during acclimation of freshwater salmon to seawater (SW) and during the smoltification process were examined. Acclimation to SW reduced...... induced no significant changes in expression of the other isoforms. This study demonstrates the expression of an array of salmon claudin isoforms and shows that SW acclimation involves inverse regulation, in the gill, of claudin 10e versus claudin 27a and 30. It is possible, that claudin 10e...

Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

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Ralph D Hector

Full Text Available Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5 cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR, which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

RON kinase isoforms demonstrate variable cell motility in normal cells.

Science.gov (United States)

Greenbaum, Alissa; Rajput, Ashwani; Wan, Guanghua

2016-09-01

Aberrant RON (Recepteur d'Origine Nantais) tyrosine kinase activation causes the epithelial cell to evade normal growth pathways, resulting in unregulated cell proliferation, increased cell motility and decreased apoptosis. Wildtype (wt) RON has been shown to play a role in metastasis of epithelial malignancies. It presents an important potential therapeutic target for colorectal, breast, gastric and pancreatic cancer. Little is known about functional differences amongst RON isoforms RON155, RON160 and RON165. The purpose of this study was to determine the effect of various RON kinase isoforms on cell motility. Cell lines with stable expression of wtRON were generated by inserting the coding region of RON in pTagRFP (tagged red fluorescence protein plasmid). The expression constructs of RON variants (RON155, RON160 and RON165) were generated by creating a mutagenesis-based wtRON-pTag RFP plasmid and stably transfected into HEK 293 cells. The wound closure scratch assay was used to investigate the effect on cell migratory capacity of wild type RON and its variants. RON transfected cells demonstrated increased cell motility compared to HEK293 control cells. RON165 cell motility was significantly increased compared to RON160 (mean percentage of wound covered 37.37% vs. 32.40%; p = 0.03). RON tyrosine kinase isoforms have variable cell motility. This may reflect a difference in the behavior of malignant epithelial cells and their capacity for metastasis.

Apolipoprotein (A) Isoform Distribution and Plasma Lipoprotein (a ...

African Journals Online (AJOL)

Plasma lipoprotein (a) Concentrations and apo(a) isoforms were determined in 101 healthy Nigerian subjects (M=63), F=38; age range 17-68 years), and coronary heart disease (CHD) patients (M=19, F=17, age range 30-79 years). Median Lp(a) level was 24.4 mg/di in the CHD patients and 22.1 mg/di in the controls.

Apo E isoforms, insulin output and plasma lipid levels in essential hypertension.

Science.gov (United States)

Dembińska-Kieć, A; Kawecka-Jaszcz, K; Kwaśniak, M; Guevara, I; Pankiewicz, J; Malczewska-Malec, M; Iwanejko, J; Hartwich, J; Zdzienicka, A; Stochmal, A; Leszczyńska-Gołabek, I

1998-02-01

The association between apo E isoforms and insulin output during the oral glucose test (OGTT) in 60 non-diabetic, non-obese patients with essential hypertension and in control subjects (non-obese, non-diabetic normotensive subjects) was estimated. According to low or high insulin output during OGTT, the subjects were divided into the following groups: normotensive subjects with low (NLI) and high (NHI) and hypertensive subjects with low (HLI) and high (HHI) insulin output. The apo E 4/2 phenotype was detected in 32% of hypertensive subjects but not in control subjects. The frequency of apo E 3/2 phenotype in hypertensive subjects was 5% and in normotensive subjects 15%. An increased frequency of phenotype apo E 4/3 was noticed both in HHI (46%) and in NHI (50%) compared with HLI (22%) and NLI (17%) groups. The results suggest that the determination of phenotypes apo E and insulin output may contribute to an early detection of individuals at high risk of hypertension development.

Multiple isoforms for the catalytic subunit of PKA in the basal fungal lineage Mucor circinelloides.

Science.gov (United States)

Fernández Núñez, Lucas; Ocampo, Josefina; Gottlieb, Alexandra M; Rossi, Silvia; Moreno, Silvia

2016-12-01

Protein kinase A (PKA) activity is involved in dimorphism of the basal fungal lineage Mucor. From the recently sequenced genome of Mucor circinelloides we could predict ten catalytic subunits of PKA. From sequence alignment and structural prediction we conclude that the catalytic core of the isoforms is conserved, and the difference between them resides in their amino termini. This high number of isoforms is maintained in the subdivision Mucoromycotina. Each paralogue, when compared to the ones form other fungi is more homologous to one of its orthologs than to its paralogs. All of these fungal isoforms cannot be included in the class I or II in which fungal protein kinases have been classified. mRNA levels for each isoform were measured during aerobic and anaerobic growth. The expression of each isoform is differential and associated to a particular growth stage. We reanalyzed the sequence of PKAC (GI 20218944), the only cloned sequence available until now for a catalytic subunit of M. circinelloides. PKAC cannot be classified as a PKA because of its difference in the conserved C-tail; it shares with PKB a conserved C2 domain in the N-terminus. No catalytic activity could be measured for this protein nor predicted bioinformatically. It can thus be classified as a pseudokinase. Its importance can not be underestimated since it is expressed at the mRNA level in different stages of growth, and its deletion is lethal. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Electrophoretic Mobility of Cardiac Myosin Heavy Chain Isoforms Revisited: Application of MALDI TOF/TOF Analysis

Czech Academy of Sciences Publication Activity Database

Arnoštová, P.; Jedelsky, P. L.; Soukup, Tomáš; Žurmanová, J.

2011-01-01

Roč. 2011, - (2011), e634253 ISSN 1110-7243 R&D Projects: GA AV ČR IAAX01110901; GA ČR(CZ) GA304/08/0256 Institutional research plan: CEZ:AV0Z50110509 Keywords : cardiac MyHC isoforms * MyHC isoform mobility * effect of thyroid hormones * mass spectrometry * SDS-PAGE and western blot Subject RIV: ED - Physiology Impact factor: 2.436, year: 2011

Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets.

Science.gov (United States)

Mangin, P H; Receveur, N; Wurtz, V; David, T; Gachet, C; Lanza, F

2009-09-01

Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib-IX complex initiates a signaling cascade leading to integrin alpha(IIb)beta(3) activation, a key process in hemostasis and thrombosis. Interaction of 14-3-3zeta with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. The aim of our study was to determine whether other members of the 14-3-3 family bind to GPIb-IX. In this study, western blot analyses showed that platelets also contain the 14-3-3beta, 14-3-3gamma, 14-3-3epsilon, 14-3-3eta and 14-3-3theta isoforms, but lack 14-3-3sigma. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14-3-3 isoforms expressed in platelets, including, as previously reported, 14-3-3zeta, bind to GPIb-IX. In addition, their interaction was found to critically require the same GPIbalpha domains (580-590 and 605-610) already identified as essential for 14-3-3zeta binding, in agreement with the conservation of the sequence of the I-helix among these different isoforms. Pull-down experiments indicated that all six 14-3-3 isoforms present in platelets bind to GPIbbeta. In contrast, deletion or mutation of the GPIbbeta intracytoplasmic tail did not affect the interaction of GPIb-IX with the 14-3-3 isoforms, questioning the importance of this domain. Our study suggests that, to inhibit GPIb-induced integrin alpha(IIb)beta(3) activation, a more appropriate strategy than inhibiting individual 14-3-3 isoforms would be to target the 14-3-3-binding motif on GPIb or, alternatively, the conserved 14-3-3 I-helix.

Analysis of Distinct Roles of CaMKK Isoforms Using STO-609-Resistant Mutants in Living Cells.

Science.gov (United States)

Fujiwara, Yuya; Hiraoka, Yuri; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

2015-06-30

To assess the isoform specificity of the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKKα mutant (Ala292Thr/Leu233Phe) and a CaMKKβ mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2-3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5'-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wild-type or the STO-609-resistant mutant of CaMKKα was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKKβ mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKKβ is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound.

Characterization of p38 MAPK isoforms for drug resistance study using systems biology approach.

Science.gov (United States)

Peng, Huiming; Peng, Tao; Wen, Jianguo; Engler, David A; Matsunami, Risë K; Su, Jing; Zhang, Le; Chang, Chung-Che Jeff; Zhou, Xiaobo

2014-07-01

p38 mitogen-activated protein kinase activation plays an important role in resistance to chemotherapeutic cytotoxic drugs in treating multiple myeloma (MM). However, how the p38 mitogen-activated protein kinase signaling pathway is involved in drug resistance, in particular the roles that the various p38 isoforms play, remains largely unknown. To explore the underlying mechanisms, we developed a novel systems biology approach by integrating liquid chromatography-mass spectrometry and reverse phase protein array data from human MM cell lines with computational pathway models in which the unknown parameters were inferred using a proposed novel algorithm called modularized factor graph. New mechanisms predicted by our models suggest that combined activation of various p38 isoforms may result in drug resistance in MM via regulating the related pathways including extracellular signal-regulated kinase (ERK) pathway and NFкB pathway. ERK pathway regulating cell growth is synergistically regulated by p38δ isoform, whereas nuclear factor kappa B (NFкB) pathway regulating cell apoptosis is synergistically regulated by p38α isoform. This finding that p38δ isoform promotes the phosphorylation of ERK1/2 in MM cells treated with bortezomib was validated by western blotting. Based on the predicted mechanisms, we further screened drug combinations in silico and found that a promising drug combination targeting ERK1/2 and NFκB might reduce the effects of drug resistance in MM cells. This study provides a framework of a systems biology approach to studying drug resistance and drug combination selection. RPPA experimental Data and Matlab source codes of modularized factor graph for parameter estimation are freely available online at http://ctsb.is.wfubmc.edu/publications/modularized-factor-graph.php. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Comparative analysis of 14-3-3 isoform expression and epigenetic alterations in colorectal cancer

International Nuclear Information System (INIS)

Young, Gavin M.; Radhakrishnan, Vijayababu M.; Centuori, Sara M.; Gomes, Cecil J.; Martinez, Jesse D.

2015-01-01

The 14-3-3 family is a group of intracellular proteins found in all eukaryotic organisms. Humans have seven isoforms that serve as scaffolds to promote interactions of regulatory phospho-proteins involved in many vital cellular processes and previous studies have shown that disturbances in native 14-3-3 levels can contribute significantly to the development of various cancers. DNA and RNA was extracted from frozen tissue samples collected by the Human Cooperative Tissue Network. RNA samples were reverse transcribed and subjected to qRT-PCR analysis using fluorescently labelled probes. Genomic DNA was treated with bisulfite and cloned into bacterial vectors for subsequent high-resolution sequencing. Mammalian NIH3T3 cells were transformed with 14-3-3 eta and Ras expression vectors synthesized from cDNA. Colonies were counted and transforming capability assessed after 21 days of growth. Cell lysates were analyzed by western blot to verify protein expression. Here we examined normal and cancerous 14-3-3 expression levels of all seven isoforms in a cohort of sporadic colorectal adenocarcinomas and in a group of tumors and their matched normals using qRT-PCR analysis. We found a statistically significant decrease in the levels of 14-3-3 sigma, eta, and zeta observed among adenocarcinomas compared to normal tissue. A parallel analysis of microarray data from the TCGA dataset confirmed that expression of sigma and eta were down-regulated in colon tumors. To explore the mechanisms behind 14-3-3 expression changes, we examined the methylation status of the sigma, eta, and zeta gene promoters in selected samples. Our data identified novel CpG methylation sites in the eta promoter consistent with epigenetic silencing of both 14-3-3 sigma and eta isoforms during colon tumorigenesis. Because epigenetic silencing is the hallmark of a tumor suppressor we tested eta in focus formation assays and found that it is capable of suppressing ras-induced transformation of NIH3T3 cells. To

Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

Science.gov (United States)

Chen, Shanyan; Meng, Fanjun; Chen, Zhenzhou; Tomlinson, Brittany N; Wesley, Jennifer M; Sun, Grace Y; Whaley-Connell, Adam T; Sowers, James R; Cui, Jiankun; Gu, Zezong

2015-01-01

Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions.

TGF-β's delay skeletal muscle progenitor cell differentiation in an isoform-independent manner

International Nuclear Information System (INIS)

Schabort, Elske J.; Merwe, Mathilde van der; Loos, Benjamin; Moore, Frances P.; Niesler, Carola U.

2009-01-01

Satellite cells are a quiescent heterogenous population of mononuclear stem and progenitor cells which, once activated, differentiate into myotubes and facilitate skeletal muscle repair or growth. The Transforming Growth Factor-β (TGF-β) superfamily members are elevated post-injury and their importance in the regulation of myogenesis and wound healing has been demonstrated both in vitro and in vivo. Most studies suggest a negative role for TGF-β on satellite cell differentiation. However, none have compared the effect of these three isoforms on myogenesis in vitro. This is despite known isoform-specific effects of TGF-β1, -β2 and -β3 on wound repair in other tissues. In the current study we compared the effect of TGF-β1, -β2 and -β3 on proliferation and differentiation of the C2C12 myoblast cell-line. We found that, irrespective of the isoform, TGF-β increased proliferation of C2C12 cells by changing the cellular localisation of PCNA to promote cell division and prevent cell cycle exit. Concomitantly, TGF-β1, -β2 and -β3 delayed myogenic commitment by increasing MyoD degradation and decreasing myogenin expression. Terminal differentiation, as measured by a decrease in myosin heavy chain (MHC) expression, was also delayed. These results demonstrate that TGF-β promotes proliferation and delays differentiation of C2C12 myoblasts in an isoform-independent manner

Frataxin mRNA Isoforms in FRDA Patients and Normal Subjects: Effect of Tocotrienol Supplementation

Directory of Open Access Journals (Sweden)

Provvidenza Maria Abruzzo

2013-01-01

Full Text Available Friedreich’s ataxia (FRDA is caused by deficient expression of the mitochondrial protein frataxin involved in the formation of iron-sulphur complexes and by consequent oxidative stress. We analysed low-dose tocotrienol supplementation effects on the expression of the three splice variant isoforms (FXN-1, FXN-2, and FXN-3 in mononuclear blood cells of FRDA patients and healthy subjects. In FRDA patients, tocotrienol leads to a specific and significant increase of FXN-3 expression while not affecting FXN-1 and FXN-2 expression. Since no structural and functional details were available for FNX-2 and FXN-3, 3D models were built. FXN-1, the canonical isoform, was then docked on the human iron-sulphur complex, and functional interactions were computed; when FXN-1 was replaced by FXN-2 or FNX-3, we found that the interactions were maintained, thus suggesting a possible biological role for both isoforms in human cells. Finally, in order to evaluate whether tocotrienol enhancement of FXN-3 was mediated by an increase in peroxisome proliferator-activated receptor-γ (PPARG, PPARG expression was evaluated. At a low dose of tocotrienol, the increase of FXN-3 expression appeared to be independent of PPARG expression. Our data show that it is possible to modulate the mRNA expression of the minor frataxin isoforms and that they may have a functional role.

Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

International Nuclear Information System (INIS)

Nemazanyy, Ivan; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T.

2006-01-01

CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy β and originally identified CoA synthase, CoASy α. The transcript specific for CoASy β was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy β. In contrast to CoASy α, which shows ubiquitous expression, CoASy β is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation

Enantioselective radical reactions. Evaluation of nitrogen protecting groups in the synthesis of β2-amino acids

Science.gov (United States)

Sibi, Mukund P.; Patil, Kalyani

2006-01-01

We have investigated the effect of nitrogen protecting groups in radical addition trapping experiments leading to β2-amino acids. Of the three N-protecting groups examined, the phthalimido group was optimal with respect to both yields and enantioselectivity. Additionally, radical additions to more complex acrylates were also investigated, which provided access to functionalized β2-amino acids in modest selectivity. PMID:16799704

Effects of Different Solvents on the Surface Acidic Oxygen-containing Functional Groups on Xanthoceras sorbifolia Shell

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Linan Liu

2014-03-01

Full Text Available This study reports the preparation of a novel biomaterial from a forestry residue - Xanthoceras sorbifolia shell (XSS - by solvent modification. The effects of acid and base (hydrochloric acerbic, acetic acid, sodium hydroxide, ammonia water and some organic solvents (ethanol, acetone, ethyl acetate, chloroform, petroleum ether, and n-hexane on the surface acidic functional groups (SAFGs on XSS were investigated. The amount of SAFGs was quantified using acid and alkali chemical titration methods, and the characteristics of virgin XSS were compared with treated ones by FT-IR spectroscopy. It was found that acid solutions can increase the concentration of SAFGs, while alkaline solutions reduce it. The XSS treated in 0.5 M HCl has the largest number of total acidic functional groups and phenolic hydroxyl groups. The shell extracted with 2 M acetic acid has the highest concentration of carboxyl. The SAFG contents were remarkably increased by treatments with ethanol and acetone, due to the outstanding enhancement of phenolic hydroxyl. These changes in the SAFGs of XSS brought about by treatments with various solutions could be a theoretical foundation for modifying this residue to create a new type of highly efficient absorbent material.

CD44 staining of cancer stem-like cells is influenced by down-regulation of CD44 variant isoforms and up-regulation of the standard CD44 isoform in the population of cells that have undergone epithelial-to-mesenchymal transition.

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Adrian Biddle

Full Text Available CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44(high cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44(high population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44(high population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44(high population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that

Kinetics of local and systemic isoforms of serum amyloid A in bovine mastitic milk

DEFF Research Database (Denmark)

Jacobsen, Stine; Niewold, T.A.; Kornalijnslijper, E.

2005-01-01

The aim of the present study was to characterise the serum amyloid A (SAA) response to intramammary inoculation of Escherichia coli and to examine the distribution of hepatically and extrahepatically pruduced SAA isoforms in plasma and milk fra cows with mastitis.......The aim of the present study was to characterise the serum amyloid A (SAA) response to intramammary inoculation of Escherichia coli and to examine the distribution of hepatically and extrahepatically pruduced SAA isoforms in plasma and milk fra cows with mastitis....

Prediction of acid dissociation constants of organic compounds using group contribution methods

DEFF Research Database (Denmark)

Zhou, Teng; Jhamb, Spardha; Liang, Xiaodong

2018-01-01

data-points with average absolute error of 0.23; (b) a non-linear GC model for organic compounds using 1622 data-points with average absolute error of 1.18; (c) an artificial neural network (ANN) based GC model for the organic compounds with average absolute error of 0.17. For each of the developed......In this paper, group contribution (GC) property models for the estimation of acid dissociation constants (Ka) of organic compounds are presented. Three GC models are developed to predict the negative logarithm of the acid dissociation constant pKa: (a) a linear GC model for amino acids using 180...

Distinct transthyretin oxidation isoform profile in spinal fluid from patients with Alzheimer’s disease and mild cognitive impairment

DEFF Research Database (Denmark)

Poulsen, Keld; Bahl, Justyna Mc; Simonsen, Anja H

2014-01-01

BACKGROUND: Transthyretin (TTR), an abundant protein in cerebrospinal fluid (CSF), contains a free, oxidation-prone cysteine residue that gives rise to TTR isoforms. These isoforms may reflect conditions in vivo. Since increased oxidative stress has been linked to neurodegenerative disorders such...

Investigation of ion exchangers with groups of aminoethylphosphonium acids and their derivatives

International Nuclear Information System (INIS)

Lejkin, Yu.A.; Ratajchak, V.; Korshak, V.V.

1977-01-01

Acid-base equilibrium has been investigated on polymeric chelate compounds with groups of aminoethylphosphonium acids, AEP-3, their monoesters, AEP-2 and diesters, AEP-1. Values of the acid-base equilibrium constants of the chelate compounds have been calculated, and schemes of consecutive protonation of the ion exchangers investigated are suggested. Sorption characteristics for the series of U, Fe, Co, Ca, Th metals have been investigated. Values of the distribution coefficients and capacity from acid-base media of various concentration are given. Assumptions are made, which explain an increase in the selectivity and affinity in the series of the chelate compounds investigated. In the AEP-3 > AEP series-2 > AEP-1 weakening of the affinity to U, Fe, Th is observed

Novel isoforms of the TFIID subunit TAF4 modulate nuclear receptor-mediated transcriptional activity

International Nuclear Information System (INIS)

Brunkhorst, Adrian; Neuman, Toomas; Hall, Anita; Arenas, Ernest; Bartfai, Tamas; Hermanson, Ola; Metsis, Madis

2004-01-01

The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF II 135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo

Skeletal muscle-specific expression of PGC-1α-b, an exercise-responsive isoform, increases exercise capacity and peak oxygen uptake.

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Miki Tadaishi

Full Text Available Maximal oxygen uptake (VO(2max predicts mortality and is associated with endurance performance. Trained subjects have a high VO(2max due to a high cardiac output and high metabolic capacity of skeletal muscles. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α, a nuclear receptor coactivator, promotes mitochondrial biogenesis, a fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training increases PGC-1α in skeletal muscle, PGC-1α-mediated changes may contribute to the improvement of exercise capacity and VO(2max. There are three isoforms of PGC-1α mRNA. PGC-1α-b protein, whose amino terminus is different from PGC-1α-a protein, is a predominant PGC-1α isoform in response to exercise. We investigated whether alterations of skeletal muscle metabolism by overexpression of PGC-1α-b in skeletal muscle, but not heart, would increase VO(2max and exercise capacity.Transgenic mice showed overexpression of PGC-1α-b protein in skeletal muscle but not in heart. Overexpression of PGC-1α-b promoted mitochondrial biogenesis 4-fold, increased the expression of fatty acid transporters, enhanced angiogenesis in skeletal muscle 1.4 to 2.7-fold, and promoted exercise capacity (expressed by maximum speed by 35% and peak oxygen uptake by 20%. Across a broad range of either the absolute exercise intensity, or the same relative exercise intensities, lipid oxidation was always higher in the transgenic mice than wild-type littermates, suggesting that lipid is the predominant fuel source for exercise in the transgenic mice. However, muscle glycogen usage during exercise was absent in the transgenic mice.Increased mitochondrial biogenesis, capillaries, and fatty acid transporters in skeletal muscles may contribute to improved exercise capacity via an increase in fatty acid utilization. Increases in PGC-1α-b protein or function might be a useful strategy for sedentary subjects to perform exercise

Progesterone receptor isoform analysis by quantitative real-time polymerase chain reaction in formalin-fixed, paraffin-embedded canine mammary dysplasias and tumors

DEFF Research Database (Denmark)

Guil-Luna, S.; Stenvang, Jan; Brünner, Nils

2014-01-01

and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign...... in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors...

Comparison of inhibition capability of scutellarein and scutellarin towards important liver UDP-glucuronosyltransferase (UGT) isoforms.

Science.gov (United States)

Ma, Guang-You; Cao, Yun-Feng; Hu, Cui-Min; Fang, Zhong-Ze; Sun, Xiao-Yu; Hong, Mo; Zhu, Zhi-Tu

2014-03-01

Scutellarin is an important bioactive flavonoid extracted from Erigeron breviscapus (Vant.) Hand-Mazz, and scutellarein is the corresponding aglycone of scutellarin. The present study aims to compare the inhibition potential of scutellarin and scutellarein towards several important UDP-glucuronosyltransferase (UGT) isoforms, including UGT1A1, UGT1A6, UGT1A9 and UGT2B7. It was demonstrated that scutellarein exerted stronger inhibition towards the tested UGT isoforms than scutellarin. Furthermore, the inhibition kinetic type and parameters (Ki ) were determined for the scutellarein's inhibition towards these UGT isoforms. Competitive inhibition of scutellarein towards all these UGT isoforms was demonstrated, and the Ki values were calculated to be 0.02, 5.0, 5.8 and 35.9 μM for UGT1A1, 1A6, 1A9 and 2B7, respectively. Using in vivo maximum plasma concentration of scutellarein in rat, the in vitro-in vivo extrapolation was performed to predict in vivo situation, indicating the most possible in vivo adverse effects due to the inhibition of scutellarein towards UGT1A1. All these results remind us to monitor the utilization of scutellarin and scutellarein, and the herbs containing these two components. Copyright © 2013 John Wiley & Sons, Ltd.

Novel causative mutations in patients with Nance-Horan syndrome and altered localization of the mutant NHS-A protein isoform

OpenAIRE

Sharma, Shiwani; Burdon, Kathryn P.; Dave, Alpana; Jamieson, Robyn V.; Yaron, Yuval; Billson, Frank; Van Maldergem, Lionel; Lorenz, Birgit; Gécz, Jozef; Craig, Jamie E.

2008-01-01

Purpose Nance-Horan syndrome is typically characterized by severe bilateral congenital cataracts and dental abnormalities. Truncating mutations in the Nance-Horan syndrome (NHS) gene cause this X-linked genetic disorder. NHS encodes two isoforms, NHS-A and NHS-1A. The ocular lens expresses NHS-A, the epithelial and neuronal cell specific isoform. The NHS-A protein localizes in the lens epithelium at the cellular periphery. The data to date suggest a role for this isoform at cell-cell junction...

The prognostic value of Her4 receptor isoform expression in triple-negative and Her2 positive breast cancer patients

International Nuclear Information System (INIS)

Machleidt, Anna; Buchholz, Stefan; Diermeier-Daucher, Simone; Zeman, Florian; Ortmann, Olaf; Brockhoff, Gero

2013-01-01

Not only four but rather seven different human epidermal growth factor receptor related (Her) receptor tyrosine kinases (RTKs) have been described to be expressed in a variety of normal and neoplastic tissues: Her1, Her2, Her3, and additionally four Her4 isoforms have been identified. A differential expression of Her4 isoforms does not, however, play any role in either the molecular diagnostics or treatment decision for breast cancer patients. The prognostic and predictive impact of Her4 expression in breast cancer is basically unclear. We quantified the Her4 variants JM-a/CYT1, JM-a/CYT2, JM-b/CYT1, and JM-b/CYT2 by isoform-specific polymerase chain reaction (qPCR) in (i) triple-negative, (ii) Her2 positive breast cancer tissues and (iii) in benign breast tissues. In all three tissue collectives we never found the JM-b/CYT1 or the JM-b/CYT2 isoform expressed. In contrast, the two JM-a/CYT1 and JM-a/CYT2 isoforms were always simultaneously expressed but at different ratios. We identified a positive prognostic impact on overall survival (OS) in triple-negative and event-free survival (EFS) in Her2 positive patients. This finding is independent of the absolute JM-a/CYT1 to JM-a/CYT2 expression ratio. In Her2 positive patients, Her4 expression only has a favorable effect in estrogen-receptor (ER)-positive but not in ER-negative individuals. In summary, JM-a/CYT1 and JM-a/CYT2 but not JM-b isoforms of the Her4 receptor are simultaneously expressed in both triple-negative and Her2 positive breast cancer tissues. Although different expression ratios of the two JM-a isoforms did not reveal any additional information, Her4 expression basically indicates a prolonged EFS and OFS. An extended expression analysis that takes all Her receptor homologs, including the Her4 isoforms, into account might render more precisely the molecular diagnostics required for the development of optimized targeted therapies

Fatty acid CoA ligase-4 gene polymorphism influences fatty acid metabolism in metabolic syndrome, but not in depression.

Science.gov (United States)

Zeman, Miroslav; Vecka, Marek; Jáchymová, Marie; Jirák, Roman; Tvrzická, Eva; Stanková, Barbora; Zák, Ales

2009-04-01

The composition of polyunsaturated fatty acids (PUFAs) in cell membranes and body tissues is altered in metabolic syndrome (MetS) and depressive disorder (DD). Within the cell, fatty acid coenzyme A (CoA) ligases (FACLs) activate PUFAs by esterifying with CoA. The FACL4 isoform prefers PUFAs (arachidonic and eicosapentaenoic acid) as substrates, and the FACL4 gene is mapped to Xq23. We have analyzed the association between the common single nucleotide polymorphism (SNP) (rs1324805, C to T substitution) in the first intron of the FACL4 gene and MetS or DD. The study included 113 healthy subjects (54 Males/59 Females), 56 MetS patients (34M/22F) and 41 DD patients (7M/34F). In MetS group, T-carriers and patients with CC or C0 (CC/C0) genotype did not differ in the values of metabolic indices of MetS and M/F ratio. Nevertheless, in comparison with CC/C0, the T-allele carriers were characterized by enhanced unfavorable changes in fatty acid metabolism typical for MetS: higher content of dihomogammalinolenic acid (P phosphatidylcholine (PC) (P = 0.052), lower index of Delta5 desaturation (P insulin, conjugated dienes and index of insulin resistance, but showed no significant association with the studied SNP. The present study shows that the common SNP (C to T substitution) in the first intron of the FACL4 gene is associated with altered FA composition of plasma phosphatidylcholines in patients with MetS.

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

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Chin-Soon Chee

2014-01-01

Full Text Available Glutathione transferases (GST were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW of 23 kDa. 2-dimensional (2-D gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5 and GST2 (pI 6.2 with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase and F0KKB0 (glutathione S-transferase III of Acinetobacter calcoaceticus strain PHEA-2, respectively.

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

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Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

2014-01-01

Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity

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Rachel S. Lee

2011-01-01

Full Text Available The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ.

Circulating soluble RAGE isoforms are attenuated in obese, impaired-glucose-tolerant individuals and are associated with the development of type 2 diabetes

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Miranda, Edwin R; Somal, Vikram S; Mey, Jacob T

2017-01-01

The soluble receptor for advanced glycation end products (sRAGE) may be protective against inflammation associated with obesity and type 2 diabetes (T2DM). The aim of this study was to determine the distribution of sRAGE isoforms and whether sRAGE isoforms are associated with risk of T2DM...... development in subjects spanning the glucose tolerance continuum. In this retrospective analysis, circulating total sRAGE and endogenous secretory RAGE (esRAGE) were quantified via ELISA, and cleaved RAGE (cRAGE) was calculated in 274 individuals stratified by glucose tolerance status (GTS) and obesity. Group......RAGE, and esRAGE were all lower with IGT and T2DM, while the ratio of cRAGE to esRAGE (cRAGE:esRAGE) was only lower (P obesity, cRAGE:esRAGE was higher with obesity and lower with IGT (P

Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats.

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Alkhedaide, Adel; Soliman, Mohamed Mohamed; Ibrahim, Zein Shaban

2016-11-01

The aim of the current study was to examine the effects of chronic consumption of soft drinks (SDs) on hepatic oxidative stress and cytochrome P450 enzymes (CYPs) expression in the livers of Wistar rats. For 3 consecutive months, the rats had free access to three different soft drinks, Coca-Cola, Pepsi-Cola and 7-UP. The rats were subsequently compared with control group rats that had consumed water. Blood and hepatic tissue samples were assayed for the changes in antioxidants, liver function biomarkers and hepatic gene expression for different isoforms of hepatic CYP. The results indicated that SD consumption (SDC) decreased serum antioxidant levels and increased malondialdehyde secretion, and increased liver biomarkers (glutamate pyruvate transaminase and glutamate oxaloacetate). SD induced alterations in mRNA expression of hepatic antioxidants and cytochrome isoforms. The expression of peroxidase, catalase, CYP1A2, CYP3A2 and CYP2C11 in the liver were upregulated following SDC. By contrast, CYP2B1 was downregulated after 3 months of SDC in liver tissue samples. Thus, the present findings indicate that SDs induced oxidative stress in the liver of Wistar rats and for the first time, to the best of our knowledge, indicate that SDC disrupts hepatic CYP enzymes that may affect drug metabolism. Therefore, drug-dosing programs should be carefully designed to take these novel findings into consideration for the treatment of diseases.

Platelet amyloid precursor protein isoform expression in Alzheimer's disease: evidence for peripheral marker.

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Vignini, A; Sartini, D; Morganti, S; Nanetti, L; Luzzi, S; Provinciali, L; Mazzanti, L; Emanuelli, M

2011-01-01

Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by a progressive cognitive and memory decline. Among peripheral markers of AD, great interest has been focused on the amyloid precursor protein (APP). In this regard, platelets represent an important peripheral source of APP since it has been demonstrated that the three major isoforms, that are constituted of 770, 751 and 695 aa residues, are inserted in the membrane of resting platelets. APP 751 and APP 770 contain a Kunitz-type serine protease inhibitor domain (APP KPI) and APP 695 lacks this domain. To address this issue, we first examined the platelet APP isoform mRNAs prospectively as biomarker for the diagnosis of AD by means of real-time quantitative PCR, and then evaluated the correlation between APP mRNA expression levels and cognitive impairment of enrolled subjects. Differential gene expression measurements in the AD patient group (n=18) revealed a significant up-regulation of APP TOT (1.52-fold), APP KPI (1.32-fold), APP 770 (1.33-fold) and APP 751 (1.26-fold) compared to controls (n=22). Moreover, a statistically significant positive correlation was found between APP mRNA levels (TOT, KPI, 770 and 751) and cognitive impairment. Since AD definitive diagnosis still relies on pathological evaluation at autopsy, the present results are consistent with the hypothesis that platelet APP could be considered a potential reliable peripheral marker for studying AD and could contribute to define a signature for the presence of AD pathology.

Lipoprotein(a) levels, apo(a) isoform size, and coronary heart disease risk in the Framingham Offspring Study

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The aim of this study was to assess the independent contributions of plasma levels of lipoprotein(a) [Lp(a)], Lp(a) cholesterol, and of apo(a) isoform size to prospective coronary heart disease (CHD) risk. Plasma Lp(a) and Lp(a) cholesterol levels, and apo(a) isoform size were measured at examinati...

A nitrous acid biosynthetic pathway for diazo group formation in bacteria.

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Sugai, Yoshinori; Katsuyama, Yohei; Ohnishi, Yasuo

2016-02-01

Although some diazo compounds have bioactivities of medicinal interest, little is known about diazo group formation in nature. Here we describe an unprecedented nitrous acid biosynthetic pathway responsible for the formation of a diazo group in the biosynthesis of the ortho-diazoquinone secondary metabolite cremeomycin in Streptomyces cremeus. This finding provides important insights into the biosynthetic pathways not only for diazo compounds but also for other naturally occurring compounds containing nitrogen-nitrogen bonds.

Cryptocephal, the Drosophila melanogaster ATF4, is a specific coactivator for ecdysone receptor isoform B2.

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Sebastien A Gauthier

Full Text Available The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR and Ultraspiracle (USP. In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4 is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here we show that Cryptocephal (CRC, the Drosophila homolog of ATF4, is an ecdysone receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote ecdysone-dependent expression of ecdysis-triggering hormone (ETH, an essential regulator of insect molting behavior. We propose that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear receptors with distinct AF1-coactivators.

The influence of the perinatal chronic hyperglycaemia on the pattern of NOS isoforms expression in left ventricle myocardium in male rats of pre-pubertal age

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O. V. Gancheva

2016-12-01

Full Text Available The aim of our study was to find out the features of the nitric oxide synthase (NOS isoforms expression in longitudinal and transversal layers of the myocardium of the left ventricle in male rats of 3 months, which are descendants of female rats with experimental gestational diabetes (EGD. Materials and methods. Study was carried out on 10 male rats descendants of female rats with normal course of pregnancy and 10 descendants of female rats with EGD. We evaluated the expression of neuronal, endothelial and inducible isoforms of NOS in histological sections both of transversal and longitudinal layers of the myocardium of left ventricle. With the aim to analyze the pattern of the NOS isoforms expression in 5 um histological slices of left ventricle myocardium we have carried out a complex of histochemical assays. Slices were allocated to 3 groups: 1st one was incubated with rabbit IgG to neuronal NOS (Santa Cruz Biotechnology, USA in dilution of 1:200; 2nd group underwent the incubation with rabbit IgG to endothelial NOS (eNos (Santa Cruz Biotechnology, USA in dilution 1:200; 3rd group was incubated with mice IgG to inducible NOS (iNos conjugated with FITC (Santa Cruz Biotechnology, USA in dilution of 1:200. The analysis of images was carried out with VIDAS-2.5 application package (Kontron Elektronik, Germany. Microimages of the left ventricle myocardium obtained with AxioScope (Carl Zeiss, Germany and COHU 4922 (COHU Inc., USA camera were processed in the system of digital image analysis VIDAS-386 (Kontron Elektronik, Germany. Results. In the study we have found that prenatal hyperglycaemia leads to the significant changes of the expression of NOS isoforms in the myocardium of left ventricle in male rats descendants of females with EGD, and the contain and the allocation of these enzymes are dependent both on type of the enzyme and its location in muscular layers. For eNOS the increase of the expression and the allocation in both transversal and

Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1

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Inserra, Marco C.; Israel, Mathilde R.; Caldwell, Ashlee; Castro, Joel; Deuis, Jennifer R.; Harrington, Andrea M.; Keramidas, Angelo; Garcia-Caraballo, Sonia; Maddern, Jessica; Erickson, Andelain; Grundy, Luke; Rychkov, Grigori Y.; Zimmermann, Katharina; Lewis, Richard J.; Brierley, Stuart M.; Vetter, Irina

2017-01-01

Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1–1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation. PMID:28225079

Heterogeneous effects of M-CSF isoforms on the progression of MLL-AF9 leukemia.

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Wang, Rong; Feng, Wenli; Yang, Feifei; Yang, Xiao; Wang, Lina; Chen, Chong; Hu, Yuting; Ren, Qian; Zheng, Guoguang

2018-02-01

Macrophage colony-stimulating factor (M-CSF) regulates both malignant cells and microenvironmental cells. Its splicing isoforms show functional heterogeneity. However, their roles on leukemia have not been well established. Here, the expression of total M-CSF in patients with hematopoietic malignancies was analyzed. The roles of M-CSF isoforms on the progression of acute myeloid leukemia (AML) were studied by establishing MLL-AF9-induced mouse AML models with high level membrane-bound M-CSF (mM-CSF) or soluble M-CSF (sM-CSF). Total M-CSF was highly expressed in myeloid leukemia patients. Furthermore, mM-CSF but not sM-CSF prolonged the survival of leukemia mice. While sM-CSF was more potent to promote proliferation and self-renew, mM-CSF was more potent to promote differentiation. Moreover, isoforms had different effects on leukemia-associated macrophages (LAMs) though they both increase monocytes/macrophages by growth-promoting and recruitment effects. In addition, mM-CSF promoted specific phagocytosis of leukemia cells by LAMs. RNA-seq analysis revealed that mM-CSF enhanced phagocytosis-associated genes and activated oxidative phosphorylation and metabolism pathway. These results highlight heterogeneous effects of M-CSF isoforms on AML progression and the mechanisms of mM-CSF, that is, intrinsically promoting AML cell differentiation and extrinsically enhancing infiltration of macrophages and phagocytosis by macrophages, which may provide potential clues for clinical diagnosis and therapy. © 2017 Australasian Society for Immunology Inc.

VEGF-A isoforms differentially regulate ATF-2-dependent VCAM-1 gene expression and endothelial-leukocyte interactions.

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Fearnley, Gareth W; Odell, Adam F; Latham, Antony M; Mughal, Nadeem A; Bruns, Alexander F; Burgoyne, Nicholas J; Homer-Vanniasinkam, Shervanthi; Zachary, Ian C; Hollstein, Monica C; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

2014-08-15

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways. © 2014 Fearnley et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Increased Expression of the Na,K-ATPase alpha4 Isoform Enhances Sperm Motility in Transgenic Mice1

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Jimenez, Tamara; Sanchez, Gladis; McDermott, Jeffrey P.; Nguyen, Anh-Nguyet; Kumar, T. Rajendra; Blanco, Gustavo

2010-01-01

The Na,K-ATPase alpha4 (ATP1A4) isoform is specifically expressed in male germ cells and is highly prevalent in spermatozoa. Although selective inhibition of alpha4 activity with ouabain has been shown to affect sperm motility, a more direct analysis of the role of this isoform in sperm movement has not yet been demonstrated. To establish this, we engineered transgenic mice that express the rat alpha4 isoform fused to green fluorescent protein in male germ cells, under the control of the mouse protamine 1 promoter. We showed that the rat Atp1a4 transgene is expressed in mouse spermatozoa and that it is localized to the sperm flagellum. In agreement with increased expression of the alpha4 isoform, sperm from transgenic mice displayed higher alpha4-specific Na,K-ATPase activity and binding of fluorescently labeled ouabain than wild-type mice. In contrast, expression and activity of ATP1A1 (alpha1), the other Na,K-ATPase alpha isoform present in sperm, remained unchanged. Similar to wild-type mice, mice expressing the alpha4 transgene exhibited normal testis and sperm morphology and no differences in fertility. However, compared to wild-type mice, sperm from transgenic mice displayed plasma membrane hyperpolarization and higher total and progressive motility. Other parameters of motility also increased, including straight-line, curvilinear, and average path velocities and amplitude of lateral head displacement. In addition, sperm from the transgenic mice showed enhanced sperm hyperactive motility, but no changes in progesterone-induced acrosome reaction. Altogether, these results provide new genetic evidence for the role of the ATP1A4 isoform in sperm motility, under both noncapacitating and capacitating conditions. PMID:20826726

Acidic surface functional groups and mineral elements in Lakra coal (Sindh, Pakistan)

International Nuclear Information System (INIS)

Saeed, K.; Ishaq, M.; Ahjmad, I.; Shakirullah, M.; Haider, S.

2010-01-01

Surface acidity of virgin coal (Lakra Sindh, Pakistan) and variously extracted/leached coal samples with HNO/sub 3/ NaOH, and KMnO/sub 4/, were investigated by aqueous potentiometric titration employing KOH as a titrant. The titration curve of virgin coal showed that its surface might contain carboxylic, carbonyl, phenolic and other weak acidic functional groups such as enols and C-H bond. The titration curves of leached coal samples showed inflections at pH 4-11, being not similar the inflections of carboxylic groups. This inflection might be given by functional groups like CO/sub 2/, phenolic, enols and C-H. Mineral matter such as Fe, K, Zn, Mn and Ni were determined in the ash of coal by atomic absorption spectrophotometer and was found that Fe (3104 micro g/g) in the highest and Ni (36.05 micro g/g) in the lowest quantity is present in virgin coal sample. (author)

Cytoplasmic isoforms of Kaposi sarcoma herpesvirus LANA recruit and antagonize the innate immune DNA sensor cGAS.

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Zhang, Guigen; Chan, Baca; Samarina, Naira; Abere, Bizunesh; Weidner-Glunde, Magdalena; Buch, Anna; Pich, Andreas; Brinkmann, Melanie M; Schulz, Thomas F

2016-02-23

The latency-associated nuclear antigen (LANA) of Kaposi sarcoma herpesvirus (KSHV) is mainly localized and functions in the nucleus of latently infected cells, playing a pivotal role in the replication and maintenance of latent viral episomal DNA. In addition, N-terminally truncated cytoplasmic isoforms of LANA, resulting from internal translation initiation, have been reported, but their function is unknown. Using coimmunoprecipitation and MS, we found the cGMP-AMP synthase (cGAS), an innate immune DNA sensor, to be a cellular interaction partner of cytoplasmic LANA isoforms. By directly binding to cGAS, LANA, and particularly, a cytoplasmic isoform, inhibit the cGAS-STING-dependent phosphorylation of TBK1 and IRF3 and thereby antagonize the cGAS-mediated restriction of KSHV lytic replication. We hypothesize that cytoplasmic forms of LANA, whose expression increases during lytic replication, inhibit cGAS to promote the reactivation of the KSHV from latency. This observation points to a novel function of the cytoplasmic isoforms of LANA during lytic replication and extends the function of LANA from its role during latency to the lytic replication cycle.

Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds.

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Troncoso-Ponce, M A; Rivoal, J; Venegas-Calerón, M; Dorion, S; Sánchez, R; Cejudo, F J; Garcés, R; Martínez-Force, E

2012-07-01

Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues. Copyright © 2012 Elsevier Ltd. All rights reserved.

Selective inhibition of sheep kidney 11 beta-hydroxysteroid dehydrogenase isoform 2 activity by 5 alpha-reduced (but not 5 beta) derivatives of adrenocorticosteroids.

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Latif, S A; Sheff, M F; Ribeiro, C E; Morris, D J

1997-02-01

We have previously reported that 5 alpha and 5 beta pathways of steroid metabolism are controlled in vivo by dietary Na+ and glycyrrhetinic acid, see Gorsline et al. 1988; Latif et al. 1990. The present investigations provide evidence supporting the suggestion that endogenous substances may regulate the glucocorticoid inactivating isoenzymes, 11 beta-HSD (hydroxysteroid dehydrogenase) 1 (liver) and 11 beta-HSD2 (kidney). The activity of 11 beta-HSD is impaired in essential hypertension, following licorice ingestion, and in patients with apparent mineralocorticoid excess where 11 beta-HSD2 is particularly affected. In all three conditions, excretion of the less common 5 alpha metabolites is elevated in urine. We now report on the differential abilities of a series of Ring A reduced (5 alpha and 5 beta) adrenocorticosteroid and progesterone metabolites to inhibit these isoenzymes. Using liver microsomes with NADP+ as co-factor (11 beta-HSD1), and sheep kidney microsomes with NAD+ as co-factor (11 beta-HSD2), we have systematically investigated the abilities of a number of adrenocorticosteroids and their derivatives to inhibit the individual isoforms of 11 beta-HSD. A striking feature is the differential sensitivity of the two isoenzymes to inhibition by 5 alpha and 5 beta derivatives. 11 beta-HSD1 is inhibited by both 5 alpha and certain 5 beta derivatives. 11 beta-HSD-2 was selectively inhibited only by 5 alpha derivatives: 5 beta derivatives were without inhibitory activity toward this isoform of 11 beta-HSD. These results indicate the importance of the structural conformation of the A and B Rings in conferring specific inhibitory properties on these compounds. In addition, we discuss the effects of additions or substitutions of other functional groups on the inhibitory potency of these steroid molecules against 11 beta-HSD1 and 11 beta-HSD2.

Cardiotonic steroids trigger non-classical testosterone signaling in Sertoli cells via the α4 isoform of the sodium pump.

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Konrad, Lutz; Dietze, Raimund; Kirch, Ulrike; Kirch, Herbert; Eva, Alexander; Scheiner-Bobis, Georgios

2011-12-01

The α4 isoform of the Na(+),K(+)-ATPase (sodium pump) is known to be expressed in spermatozoa and to be critical for their motility. In the investigation presented here, we find that the rat-derived Sertoli cell line 93RS2 also expresses considerable amounts of the α4 isoform in addition to the α1 isoform. Since Sertoli cells are not motile, one can assume that the function of the α4 isoform in these cells must differ from that in spermatozoa. Thus, we assessed a potential involvement of this isoform in signaling pathways that are activated by the cardiotonic steroid (CTS) ouabain, a highly specific sodium pump ligand. Treatment of 93RS2 cells with ouabain leads to activation of the c-Src/c-Raf/Erk1/2 signaling cascade. Furthermore, we show for the first time that the activation of this cascade by ouabain results in phosphorylation and activation of the transcription factor CREB. This signaling cascade is induced at low nanomolar concentrations of ouabain, consistent with the involvement of the α4 isoform. This is further supported by experiments involving siRNA: silencing of α4 expression entirely blocks ouabain-induced activation of Erk1/2 whereas silencing of α1 has no effect. The findings of this study unveil new aspects in CTS/sodium pump interactions by demonstrating for the first time ouabain-induced signaling through the α4 isoform. The c-Src/c-Raf/Erk1/2/CREB cascade activated by ouabain is identical to the so-called non-classical signaling cascade that is normally triggered in Sertoli cells by testosterone. Taking into consideration that CTS are produced endogenously, our results may help to gain new insights into the physiological mechanisms associated with male fertility and reproduction. Copyright © 2011 Elsevier B.V. All rights reserved.

The Regulation of Nitric Oxide Synthase Isoform Expression in Mouse and Human Fallopian Tubes: Potential Insights for Ectopic Pregnancy

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Junting Hu

2014-12-01

Full Text Available Nitric oxide (NO is highly unstable and has a half-life of seconds in buffer solutions. It is synthesized by NO-synthase (NOS, which has been found to exist in the following three isoforms: neuro nitric oxide synthase (nNOS, inducible nitric oxide synthase (iNOS, and endothelial nitric oxide synthase (eNOS. NOS activity is localized in the reproductive tracts of many species, although direct evidence for NOS isoforms in the Fallopian tubes of mice is still lacking. In the present study, we investigated the expression and regulation of NOS isoforms in the mouse and human Fallopian tubes during the estrous and menstrual cycles, respectively. We also measured isoform expression in humans with ectopic pregnancy and in mice treated with lipopolysaccharide (LPS. Our results confirmed the presence of different NOS isoforms in the mouse and human Fallopian tubes during different stages of the estrous and menstrual cycles and showed that iNOS expression increased in the Fallopian tubes of women with ectopic pregnancy and in LPS-treated mice. Elevated iNOS activity might influence ovulation, cilia beats, contractility, and embryo transportation in such a manner as to increase the risk of ectopic pregnancy. This study has provided morphological and molecular evidence that NOS isoforms are present and active in the human and mouse Fallopian tubes and suggests that iNOS might play an important role in both the reproductive cycle and infection-induced ectopic pregnancies.

Design, Synthesis and Biological Evaluation of Histone Deacetylase (HDAC) Inhibitors: Saha (Vorinostat) Analogs and Biaryl Indolyl Benzamide Inhibitors Display Isoform Selectivity

Science.gov (United States)

Negmeldin, Ahmed Thabet

HDAC proteins have emerged as interesting targets for anti-cancer drugs due to their involvement in cancers, as well as several other diseases. Several HDAC inhibitors have been approved by the FDA as anti-cancer drugs, including SAHA (suberoylanilide hydroxamic acid, Vorinostat). Unfortunately, SAHA inhibits most HDAC isoforms, which limit its use as a pharmacological tool and may lead to side effects in the clinic. In this work we were interested in developing isoform selective HDAC inhibitors, which may decrease or eliminate the side effects associated with non-selective inhibitors treatment. In addition, isoform selective HDAC inhibitors can be used as biological tools to help understand the HDAC-related cancer biology. Our strategy was based on synthesis and screening of several derivatives of the non-selective FDA approved drug SAHA substituted at different positions of the linker region. Several SAHA analogs modified at the C4 and C5 positions of the linker were synthesized. The new C4- and C5-modified SAHA libraries, along with the previously synthesized C2-modified SAHA analogs were screened in vitro and in cellulo for HDAC isoform selectivity. Interestingly, several analogs exhibited dual HDAC6/HDAC8 selectivity. Enantioselective syntheses of the pure enantiomers of some of the interesting analogs were performed and the enantiomers were screened in vitro. Among the most interesting analogs, ( R)-C4-benzyl SAHA displayed 520- to 1300-fold selectivity for HDAC6 and HDAC8 over HDAC1, 2, and 3, with IC50 values of 48 and 27 nM with HDAC6 and 8, respectively. Docking studies were performed to provide structural rationale for the observed selectivity of the new analogs. In addition, rational design, synthesis, and screening of several other biaryl indolyl benzamide HDAC inhibitors is discussed, and some showed modest HDAC1 selectivity. The new biaryl indolyl benzamides can be useful to further develop HDAC1 selective inhibitors. The dual HDAC6/8 selective

Serum amyloid A isoforms in serum and synovial fluid from spontaneously diseased dogs with joint diseases or other conditions

DEFF Research Database (Denmark)

Kjelgaard-Hansen, Mads Jens; Christensen, Michelle B.; Lee, Marcel Huisung

2007-01-01

Serum amyloid A (SAA) is a major acute phase protein in dogs. However, knowledge of qualitative properties of canine SAA and extent of its synthesis in extrahepatic tissues is limited. The aim of the study was to investigate expression of different SAA isoforms in serum and synovial fluid...... in samples obtained from dogs (n = 16) suffering from different inflammatory or non-inflammatory conditions, which were either related or unrelated to joints. Expression of SAA isoforms was visualized by denaturing isoelectric focusing and Western blotting. Serum amyloid A was present in serum from all dogs...... with systemic inflammatory activity, and up to four major isoforms with apparent isoelectric points between 6.1 and 7.9 were identified. In synovial fluid from inflamed joints one or more highly alkaline SAA isoforms (with apparent isoelectric points above 9.3) were identified, with data suggesting local...

Complex p63 mRNA isoform expression patterns in squamous cell carcinoma of the head and neck

DEFF Research Database (Denmark)

Thurfjell, N.; Coates, P.J.; Uusitalo, T.

2004-01-01

on the role of p63 expression in human tumours, we used quantitative real-time RT-PCR to study individual p63 isoforms in squamous cell carcinomas of the head and neck (SCCHN). In keeping with previous reports, expression of the deltaN- and p63alpha-isoforms predominated and deltaNp63 mRNA was expressed...

Entropy-based model for miRNA isoform analysis.

Directory of Open Access Journals (Sweden)

Shengqin Wang

Full Text Available MiRNAs have been widely studied due to their important post-transcriptional regulatory roles in gene expression. Many reports have demonstrated the evidence of miRNA isoform products (isomiRs in high-throughput small RNA sequencing data. However, the biological function involved in these molecules is still not well investigated. Here, we developed a Shannon entropy-based model to estimate isomiR expression profiles of high-throughput small RNA sequencing data extracted from miRBase webserver. By using the Kolmogorov-Smirnov statistical test (KS test, we demonstrated that the 5p and 3p miRNAs present more variants than the single arm miRNAs. We also found that the isomiR variant, except the 3' isomiR variant, is strongly correlated with Minimum Free Energy (MFE of pre-miRNA, suggesting the intrinsic feature of pre-miRNA should be one of the important factors for the miRNA regulation. The functional enrichment analysis showed that the miRNAs with high variation, particularly the 5' end variation, are enriched in a set of critical functions, supporting these molecules should not be randomly produced. Our results provide a probabilistic framework for miRNA isoforms analysis, and give functional insights into pre-miRNA processing.

Heterogeneity of serum gelatinases MMP-2 and MMP-9 isoforms and charge variants

Science.gov (United States)

Rossano, Rocco; Larocca, Marilena; Riviello, Lea; Coniglio, Maria Gabriella; Vandooren, Jennifer; Liuzzi, Grazia Maria; Opdenakker, Ghislain; Riccio, Paolo

2014-01-01

The matrix metalloproteinases (MMPs) gelatinase A (MMP-2) and gelatinase B (MMP-9) are mediators of brain injury in multiple sclerosis (MS) and valuable biomarkers of disease activity. We applied bidimensional zymography (2-DZ) as an extension of classic monodimensional zymography (1-DZ) to analyse the complete pattern of isoforms and post-translational modifications of both MMP-9 and MMP-2 present in the sera of MS patients. The enzymes were separated on the basis of their isoelectric points (pI) and apparent molecular weights (Mw) and identified both by comparison with standard enzyme preparations and by Western blot analysis. Two MMP-2 isoforms, and at least three different isoforms and two different states of organization of MMP-9 (the multimeric MMP-9 and the N-GAL-MMP-9 complex) were observed. In addition, 2-DZ revealed for the first time that all MMP-9 and MMP-2 isoforms actually exist in the form of charge variants: four or five variants in the N-GAL complex, more charge variants in the case of MMP-9; and five to seven charge variants for MMP-2. Charge variants were also observed in recombinant enzymes and, after concentration, also in sera from healthy individuals. Sialylation (MMP-9) and phosphorylation (MMP-2) contributed to molecular heterogeneity. The detection of charge variants of MMP-9 and MMP-2 in MS serum samples illustrates the power of 2-DZ and demonstrates that in previous studies MMP mixtures, rather than single molecules, were analysed. These observations open perspectives for better diagnosis and prognosis of many diseases and need to be critically interpreted when applying other methods for MS and other diseases. PMID:24616914

Endogenous Isoquinoline Alkaloids Agonists of Acid-Sensing Ion Channel Type 3

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Dmitry I. Osmakov

2017-09-01

Full Text Available Acid-sensing ion channels (ASICs ASIC3 expressed mainly in peripheral sensory neurons play an important role in pain perception and inflammation development. In response to acidic stimuli, they can generate a unique biphasic current. At physiological pH 7.4, human ASIC3 isoform (hASIC3 is desensitized and able to generate only a sustained current. We found endogenous isoquinoline alkaloids (EIAs, which restore hASIC3 from desensitization and recover the transient component of the current. Similarly, rat ASIC3 isoform (rASIC3 can also be restored from desensitization (at pH < 7.0 by EIAs with the same potency. At physiological pH and above, EIAs at high concentrations were able to effectively activate hASIC3 and rASIC3. Thus, we found first endogenous agonists of ASIC3 channels that could both activate and prevent or reverse desensitization of the channel. The decrease of EIA levels could be suggested as a novel therapeutic strategy for treatment of pain and inflammation.

Immunopositivity for histone macroH2A1 isoforms marks steatosis-associated hepatocellular carcinoma.

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Francesca Rappa

Full Text Available Hepatocellular carcinoma (HCC is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously.We examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fat-associated HCC, the high fat diet/diethylnistrosamine (DEN and the phosphatase and tensin homolog (PTEN liver specific knock-out (KO mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry.Protein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p<0.01 in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2, when compared to steatosis (<2% of hepatocytes positive for either isoform. The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2.These data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human HCC.

The novel protein kinase C epsilon isoform modulates acetylcholine release in the rat neuromuscular junction.

Science.gov (United States)

Obis, Teresa; Hurtado, Erica; Nadal, Laura; Tomàs, Marta; Priego, Mercedes; Simon, Anna; Garcia, Neus; Santafe, Manel M; Lanuza, Maria A; Tomàs, Josep

2015-12-01

Various protein kinase C (PKC) isoforms contribute to the phosphorylating activity that modulates neurotransmitter release. In previous studies we showed that nPKCε is confined in the presynaptic site of the neuromuscular junction and its presynaptic function is activity-dependent. Furthermore, nPKCε regulates phorbol ester-induced acetylcholine release potentiation, which further indicates that nPKCε is involved in neurotransmission. The present study is designed to examine the nPKCε involvement in transmitter release at the neuromuscular junction. We use the specific nPKCε translocation inhibitor peptide εV1-2 and electrophysiological experiments to investigate the involvement of this isoform in acetylcholine release. We observed that nPKCε membrane translocation is key to the synaptic potentiation of NMJ, being involved in several conditions that upregulate PKC isoforms coupling to acetylcholine (ACh) release (incubation with high Ca(2+), stimulation with phorbol esters and protein kinase A, stimulation with adenosine 3',5'-cyclic monophosphorothioate, 8-Bromo-, Rp-isomer, sodium salt -Sp-8-BrcAMP-). In all these conditions, preincubation with the nPKCε translocation inhibitor peptide (εV1-2) impairs PKC coupling to acetylcholine release potentiation. In addition, the inhibition of nPKCε translocation and therefore its activity impedes that presynaptic muscarinic autoreceptors and adenosine autoreceptors modulate transmitter secretion. Together, these results point to the importance of nPKCε isoform in the control of acetylcholine release in the neuromuscular junction.

Cloning of a neonatal calcium atpase isoform (SERCA 1B) from extraocular muscle of adult blue marlin (Makaira nigricans).

Science.gov (United States)

Londraville, R L; Cramer, T D; Franck, J P; Tullis, A; Block, B A

2000-10-01

Complete cDNAs for the fast-twitch Ca2+ -ATPase isoform (SERCA 1) were cloned and sequenced from blue marlin (Makaira nigricans) extraocular muscle (EOM). Complete cDNAs for SERCA 1 were also cloned from fast-twitch skeletal muscle of the same species. The two sequences are identical over the coding region except for the last five codons on the carboxyl end; EOM SERCA 1 cDNA codes for 996 amino acids and the fast-twitch cDNAs code for 991 aa. Phylogenetic analysis revealed that EOM SERCA 1 clusters with an isoform of Ca2+ -ATPase normally expressed in early development of mammals (SERCA 1B). This is the first report of SERCA 1B in an adult vertebrate. RNA hybridization assays indicate that 1B expression is limited to extraocular muscles. Because EOM gives rise to the thermogenic heater organ in marlin, we investigated whether SERCA 1B may play a role in heat generation, or if 1B expression is common in EOM among vertebrates. Chicken also expresses SERCA 1B in EOM, but rat expresses SERCA 1A; because SERCA 1B is not specific to heater tissue we conclude it is unlikely that it plays a specific role in intracellular heat production. Comparative sequence analysis does reveal, however, several sites that may be the source of functional differences between fish and mammalian SERCAs.

Solving nucleic acid structures by molecular replacement: examples from group II intron studies

International Nuclear Information System (INIS)

Marcia, Marco; Humphris-Narayanan, Elisabeth; Keating, Kevin S.; Somarowthu, Srinivas; Rajashankar, Kanagalaghatta; Pyle, Anna Marie

2013-01-01

Strategies for phasing nucleic acid structures by molecular replacement, using both experimental and de novo designed models, are discussed. Structured RNA molecules are key players in ensuring cellular viability. It is now emerging that, like proteins, the functions of many nucleic acids are dictated by their tertiary folds. At the same time, the number of known crystal structures of nucleic acids is also increasing rapidly. In this context, molecular replacement will become an increasingly useful technique for phasing nucleic acid crystallographic data in the near future. Here, strategies to select, create and refine molecular-replacement search models for nucleic acids are discussed. Using examples taken primarily from research on group II introns, it is shown that nucleic acids are amenable to different and potentially more flexible and sophisticated molecular-replacement searches than proteins. These observations specifically aim to encourage future crystallographic studies on the newly discovered repertoire of noncoding transcripts

Facile fabrication of siloxane @ poly (methylacrylic acid) core-shell microparticles with different functional groups

Energy Technology Data Exchange (ETDEWEB)

Zhao, Zheng-Bai; Tai, Li; Zhang, Da-Ming; Jiang, Yong, E-mail: yj@seu.edu.cn [Southeast University, School of Chemistry and Chemical Engineering (China)

2017-02-15

Siloxane @ poly (methylacrylic acid) core-shell microparticles with functional groups were prepared by a facile hydrolysis-condensation method in this work. Three different silane coupling agents 3-methacryloxypropyltrimethoxysilane (MPS), 3-triethoxysilylpropylamine (APTES), and 3-glycidoxypropyltrimethoxysilane (GPTMS) were added along with tetraethoxysilane (TEOS) into the polymethylacrylic acid (PMAA) microparticle ethanol dispersion to form the Si@PMAA core-shell microparticles with different functional groups. The core-shell structure and the surface special functional groups of the resulting microparticles were measured by transmission electron microscopy and FTIR. The sizes of these core-shell microparticles were about 350–400 nm. The corresponding preparation conditions and mechanism were discussed in detail. This hydrolysis-condensation method also could be used to functionalize other microparticles which contain active groups on the surface. Meanwhile, the Si@PMAA core-shell microparticles with carbon-carbon double bonds and amino groups have further been applied to prepare hydrophobic coatings.

Facile fabrication of siloxane @ poly (methylacrylic acid) core-shell microparticles with different functional groups

International Nuclear Information System (INIS)

Zhao, Zheng-Bai; Tai, Li; Zhang, Da-Ming; Jiang, Yong

2017-01-01

Siloxane @ poly (methylacrylic acid) core-shell microparticles with functional groups were prepared by a facile hydrolysis-condensation method in this work. Three different silane coupling agents 3-methacryloxypropyltrimethoxysilane (MPS), 3-triethoxysilylpropylamine (APTES), and 3-glycidoxypropyltrimethoxysilane (GPTMS) were added along with tetraethoxysilane (TEOS) into the polymethylacrylic acid (PMAA) microparticle ethanol dispersion to form the Si@PMAA core-shell microparticles with different functional groups. The core-shell structure and the surface special functional groups of the resulting microparticles were measured by transmission electron microscopy and FTIR. The sizes of these core-shell microparticles were about 350–400 nm. The corresponding preparation conditions and mechanism were discussed in detail. This hydrolysis-condensation method also could be used to functionalize other microparticles which contain active groups on the surface. Meanwhile, the Si@PMAA core-shell microparticles with carbon-carbon double bonds and amino groups have further been applied to prepare hydrophobic coatings.

Measuring the concentration of carboxylic acid groups in torrefied spruce wood.

Science.gov (United States)

Khazraie Shoulaifar, Tooran; Demartini, Nikolai; Ivaska, Ari; Fardim, Pedro; Hupa, Mikko

2012-11-01

Torrefaction is moderate thermal treatment (∼200-300°C) to improve the energy density, handling and storage properties of biomass fuels. In biomass, carboxylic sites are partially responsible for its hygroscopic. These sites are degraded to varying extents during torrefaction. In this paper, we apply methylene blue sorption and potentiometric titration to measure the concentration of carboxylic acid groups in spruce wood torrefied for 30min at temperatures between 180 and 300°C. The results from both methods were applicable and the values agreed well. A decrease in the equilibrium moisture content at different humidity was also measured for the torrefied wood samples, which is in good agreement with the decrease in carboxylic acid sites. Thus both methods offer a means of directly measuring the decomposition of carboxylic groups in biomass during torrefaction as a valuable parameter in evaluating the extent of torrefaction which provides new information to the chemical changes occurring during torrefaction. Copyright © 2012 Elsevier Ltd. All rights reserved.

SSP: an interval integer linear programming for de novo transcriptome assembly and isoform discovery of RNA-seq reads.

Science.gov (United States)

Safikhani, Zhaleh; Sadeghi, Mehdi; Pezeshk, Hamid; Eslahchi, Changiz

2013-01-01

Recent advances in the sequencing technologies have provided a handful of RNA-seq datasets for transcriptome analysis. However, reconstruction of full-length isoforms and estimation of the expression level of transcripts with a low cost are challenging tasks. We propose a novel de novo method named SSP that incorporates interval integer linear programming to resolve alternatively spliced isoforms and reconstruct the whole transcriptome from short reads. Experimental results show that SSP is fast and precise in determining different alternatively spliced isoforms along with the estimation of reconstructed transcript abundances. The SSP software package is available at http://www.bioinf.cs.ipm.ir/software/ssp. © 2013.

Nitric oxide synthase isoforms in spontaneous and salt hypertension

Czech Academy of Sciences Publication Activity Database

Hojná, Silvie; Kuneš, Jaroslav; Zicha, Josef

2007-01-01

Roč. 25, Suppl. 2 (2007), S 338-S 338 ISSN 0263-6352. [European Meeting on Hypertension /17./. 15.06.2007-19.06.2007, Milan] R&D Projects: GA MŠk(CZ) 1M0510 Institutional research plan: CEZ:AV0Z50110509 Keywords : nitric oxide synthase isoforms * spontaneous and salt hypertension Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery

Analysis of a FANCE Splice Isoform in Regard to DNA Repair.

Science.gov (United States)

Bouffard, Frédérick; Plourde, Karine; Bélanger, Simon; Ouellette, Geneviève; Labrie, Yvan; Durocher, Francine

2015-09-25

The FANC-BRCA DNA repair pathway is activated in response to interstrand crosslinks formed in DNA. A homozygous mutation in 1 of the 17 Fanconi anemia (FA) genes results in malfunctions of this pathway and development of FA syndrome. The integrity of this protein network is essential for good maintenance of DNA repair process and genome stability. Following the identification of an alternatively splice isoform of FANCE (Fanconi anemia complementation group E) significantly expressed in breast cancer individuals from high-risk non-BRCA1/2 families, we studied the impact of this FANCE splice isoform (FANCEΔ4) on DNA repair processes. We have demonstrated that FANCEΔ4 mRNA was efficiently translated into a functional protein and expressed in normal and breast cancer cell lines. Following treatment with the crosslinking agent mitomycin C, EUFA130 (FANCE-deficient) cells infected with FANCEΔ4 were blocked into G2/M phase, while cell survival was significantly reduced compared with FANCE-infected EUFA130 cells. In addition, FANCEΔ4 did not allow FANCD2 and FANCI monoubiquitination, which represents a crucial step of the FANC-BRCA functional pathway. As observed for FANCE wild-type protein, localization of FANCEΔ4 protein was confined to the nucleus following mitomycin C treatment. Although FANCEΔ4 protein showed interaction with FANCE, FANCEΔ4 did not support normal function of FANCE protein in this pathway and could have deleterious effects on FANCE protein activity. We have demonstrated that FANCEΔ4 seems to act as a regulator of FANCD2 protein expression level by promoting its degradation. This study highlights the importance of an efficient regulation of alternative splicing expression of FA genes for proper DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

Muscle-Type Specific Autophosphorylation of CaMKII Isoforms after Paced Contractions

NARCIS (Netherlands)

Eilers, W.; Gevers, W.; van Overbeek, D.; de Haan, A.; Jaspers, R.T.; Hilbers, P.A.; van Riel, A.C.R.; Flueck, M.

2014-01-01

We explored to what extent isoforms of the regulator of excitation-contraction and excitation-transcription coupling, calcium/calmodulin protein kinase II (CaMKII) contribute to the specificity of myocellular calcium sensing between muscle types and whether concentration transients in its

DIPA-family coiled-coils bind conserved isoform-specific head domain of p120-catenin family: potential roles in hydrocephalus and heterotopia.

Science.gov (United States)

Markham, Nicholas O; Doll, Caleb A; Dohn, Michael R; Miller, Rachel K; Yu, Huapeng; Coffey, Robert J; McCrea, Pierre D; Gamse, Joshua T; Reynolds, Albert B

2014-09-01

p120-catenin (p120) modulates adherens junction (AJ) dynamics by controlling the stability of classical cadherins. Among all p120 isoforms, p120-3A and p120-1A are the most prevalent. Both stabilize cadherins, but p120-3A is preferred in epithelia, whereas p120-1A takes precedence in neurons, fibroblasts, and macrophages. During epithelial-to-mesenchymal transition, E- to N-cadherin switching coincides with p120-3A to -1A alternative splicing. These isoforms differ by a 101-amino acid "head domain" comprising the p120-1A N-terminus. Although its exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here we identified delta-interacting protein A (DIPA) as the first head domain-specific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (Ccdc85a, Ccdc85b/DIPA, and Ccdc85c) interact reciprocally with p120 family members (p120, δ-catenin, p0071, and ARVCF), suggesting significant functional overlap. During zebrafish neural tube development, both knockdown and overexpression of DIPA phenocopy N-cadherin mutations, an effect bearing functional ties to a reported mouse hydrocephalus phenotype associated with Ccdc85c. These studies identify a novel, highly conserved interaction between two protein families that may participate either individually or collectively in N-cadherin-mediated development. © 2014 Markham et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

The Impact of Endurance Training on Human Skeletal Muscle Memory, Global Isoform Expression and Novel Transcripts.

Directory of Open Access Journals (Sweden)

Maléne E Lindholm

2016-09-01

Full Text Available Regularly performed endurance training has many beneficial effects on health and skeletal muscle function, and can be used to prevent and treat common diseases e.g. cardiovascular disease, type II diabetes and obesity. The molecular adaptation mechanisms regulating these effects are incompletely understood. To date, global transcriptome changes in skeletal muscles have been studied at the gene level only. Therefore, global isoform expression changes following exercise training in humans are unknown. Also, the effects of repeated interventions on transcriptional memory or training response have not been studied before. In this study, 23 individuals trained one leg for three months. Nine months later, 12 of the same subjects trained both legs in a second training period. Skeletal muscle biopsies were obtained from both legs before and after both training periods. RNA sequencing analysis of all 119 skeletal muscle biopsies showed that training altered the expression of 3,404 gene isoforms, mainly associated with oxidative ATP production. Fifty-four genes had isoforms that changed in opposite directions. Training altered expression of 34 novel transcripts, all with protein-coding potential. After nine months of detraining, no training-induced transcriptome differences were detected between the previously trained and untrained legs. Although there were several differences in the physiological and transcriptional responses to repeated training, no coherent evidence of an endurance training induced transcriptional skeletal muscle memory was found. This human lifestyle intervention induced differential expression of thousands of isoforms and several transcripts from unannotated regions of the genome. It is likely that the observed isoform expression changes reflect adaptational mechanisms and processes that provide the functional and health benefits of regular physical activity.

Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

Directory of Open Access Journals (Sweden)

Kim Dong Seon

2012-11-01

Full Text Available Abstract Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

Fish populations in a large group of acid-stressed lakes

Energy Technology Data Exchange (ETDEWEB)

Harvey, H H

1975-01-01

The purpose of this study was to determine the effects of environmental stress on the number and diversity of fish species in a group of acid-stressed lakes. The study area was the La Cloche Mountains, a series of quartzite ridges covering 1,300 km/sup 2/ along the north shore of Georgian Bay and north channel of Lake Huron. Within these ridges are 173 lakes; 68 of the largest of these made up the study sample. The lakes of the La Cloche Mountains are undergoing rapid acidification. Coincident with this there has been the loss of sport fishes from several lakes. Lakes such as Nellie, Lumsden, O.S.A., Acid and Killarney supported good sport fisheries for the lake trout, (Salvelinus namaycush) for many years, but have ceased to do so in the last 5 to 15 years. Other sport fishes, notably the walleye (Stizostedion vitreum) and smallmouth bass (micropterus dolomieu) have disappeared from some of the La Cloche Lakes. Thus recreational fishing alone could not have been the cause of the change. Beamish (1974) recorded the extreme sparcity of the three remaining fish species in O.S.A. Lake. Many of the lakes of the La Cloche mountains are accessible only with difficulty and little or no information exists for these lakes prior to this study. This precluded simple comparison of these lakes before and during acidification. This lack of historic data determined in part the approach taken in this study; a comparison of the fish communities of a group of lakes differing in degree of acid stress.

Determining the impact of oxidation on the motility of single muscle-fibres expressing different myosin isoforms

DEFF Research Database (Denmark)

Spanos, Dimitrios; Li, M.; Baron, Caroline P.

2013-01-01

heavy chain (MyHC) isoforms has not been previously investigated. Oxidation of myosin isolated from muscle fibres originating from various porcine muscles with a different metabolic profile was studied using a single muscle fibre in-vitro motility assay, allowing measurements of catalytic properties...... (motility speed) and force-generation capacity of specific MyHC isoforms. In the experimental procedure, single muscle fibres were split in different segments and each segment was exposed to a different concentration of hydrogen peroxide. Speed and force measurements were recorded and compared, to assess...... the effect of myosin oxidation on motility and force. The MyHC isoform expression in the single muscle fibre was subsequently determined on silver-stained gel SDS-PAGE. Preliminary results indicate a decrease of directionality and speed of the in-vitro motility as a result of an oxidative environment...

In vitro inhibitory activities of the extract of Hibiscus sabdariffa L. (family Malvaceae) on selected cytochrome P450 isoforms.

Science.gov (United States)

Johnson, Showande Segun; Oyelola, Fakeye Titilayo; Ari, Tolonen; Juho, Hokkanen

2013-01-01

Literature is scanty on the interaction potential of Hibiscus sabdariffa L., plant extract with other drugs and the affected targets. This study was conducted to investigate the cytochrome P450 (CYP) isoforms that are inhibited by the extract of Hibiscus sabdariffa L. in vitro. The inhibition towards the major drug metabolizing CYP isoforms by the plant extract were estimated in human liver microsomal incubations, by monitoring the CYP-specific model reactions through previously validated N-in-one assay method. The ethanolic extract of Hibiscus sabdariffa showed inhibitory activities against nine selected CYP isoforms: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. The concentrations of the extract which produced 50% inhibition of the CYP isoforms ranged from 306 µg/ml to 1660 µg/ml, and the degree of inhibition based on the IC50 values for each CYP isoform was in the following order: CYP1A2 > CYP2C8 > CYP2D6 > CYP2B6 > CYP2E1 > CYP2C19 > CYP3A4 > CYP2C9 > CYP2A6. Ethanolic extract of Hibiscus sabdariffa caused inhibition of CYP isoforms in vitro. These observed inhibitions may not cause clinically significant herb-drug interactions; however, caution may need to be taken in co-administering the water extract of Hibiscus sabdariffa with other drugs until clinical studies are available to further clarify these findings.

Ammonia IRMS-TPD measurements and DFT calculation on acidic hydroxyl groups in CHA-type zeolites.

Science.gov (United States)

Suzuki, Katsuki; Sastre, German; Katada, Naonobu; Niwa, Miki

2007-12-07

Brønsted acidity of H-chabazite (CHA) zeolites (Si : Al(2) = 4.2) was investigated by means of ammonia infrared-mass spectrometry/temperature-programmed desorption (IRMS-TPD) methods and density functional calculations. Four IR bands were observed at 3644, 3616, 3575 and 3538 cm(-1), and they were ascribable to the acidic OH groups on four nonequivalent oxygen sites in the CHA structure. The absorption band at 3538 cm(-1) was attributed to the O(4)H in the 6-membered ring (MR), and ammonia adsorption energy (DeltaU) of this OH group was the lowest among the 4 kinds of OH groups. The other 3 bands were assigned to the acidic OH groups in 8MR. It was observed that the DeltaU in 8 and 6MR were 131 (+/-3) and 101 kJ mol(-1), respectively. On the other hand, the density functional theory (DFT) calculations within periodic boundary conditions yielded the adsorption energies on these OH groups in 8 and 6MR to be ca. 130 and 110 kJ mol(-1), respectively, in good agreement with the experimentally-observed values.

Regulation of C/EBPβ isoforms by MAPK pathways in HL60 cells induced to differentiate by 1,25-dihydroxyvitamin D3

International Nuclear Information System (INIS)

Marcinkowska, Ewa; Garay, Edward; Gocek, Elzbieta; Chrobak, Agnieszka; Wang, Xuening; Studzinski, George P.

2006-01-01

C/EBPβ is known to be important for monocytic differentiation and macrophage function. Here, we found that expression of all three C/EBPβ isoforms induced in HL60 cells by 1,25-dihydroxyvitamin D 3 (1,25D) was upregulated in a sustained manner that correlates with the appearance of monocytic phenotype and with the G1 phase cell cycle arrest. In 1,25D-resistant HL60-40AF cells, isoforms β-1 and β-3 were expressed at levels comparable to 1,25D-sensitive HL60-G cells, but isoform β-2 was difficult to detect. Treatment of sensitive HL60 cells with 1,25D resulted in predominantly nuclear localization of C/EBP isoforms β-2 and β-3, while a large proportion of C/EBPβ-1 remained in the cytoplasm. Attenuation of the MEK-ERK MAPK pathway by the inhibitor PD98059 markedly reduced the expression, 1,25D-induced phosphorylation and nuclear localization of C/EBPβ-2 and C/EBPβ-3. Interestingly, only the lower molecular mass isoforms of C/EBPβ phosphorylated on Thr235 were found in the nuclei, while C/EBPβ-1 was constitutively phosphorylated and was detected principally in the cytoplasmic fraction. Although the role of C/EBPβ isoforms in 1,25D-induced differentiation is complex, our results taken together strongly suggest that the phosphorylation of C/EBPβ isoforms on Thr235 takes place mainly via the MEK-ERK pathway and that C/EBPβ-2 is the principal transcription factor in this cell system

The Effect of Carnitine Supplementation on Hyperammonemia and Carnitine Deficiency Treated with Valproic Acid in a Psychiatric Setting

OpenAIRE

Nakamura, Masaru; Nagamine, Takahiko

2015-01-01

Objective: The aim of this study was to investigate the effect of levocarnitine (active isoform of carnitine, L-Carnitine) supplementation on serum ammonia and carnitine levels simultaneously, and their clinical outcomes in valproic acid-treated psychiatric subjects.

Determinants of Isoform-Specific Gating Kinetics of hERG1 Channel: Combined Experimental and Simulation Study

Directory of Open Access Journals (Sweden)

Laura L. Perissinotti

2018-04-01

Full Text Available IKr is the rapidly activating component of the delayed rectifier potassium current, the ion current largely responsible for the repolarization of the cardiac action potential. Inherited forms of long QT syndrome (LQTS (Lees-Miller et al., 1997 in humans are linked to functional modifications in the Kv11.1 (hERG ion channel and potentially life threatening arrhythmias. There is little doubt now that hERG-related component of IKr in the heart depends on the tetrameric (homo- or hetero- channels formed by two alternatively processed isoforms of hERG, termed hERG1a and hERG1b. Isoform composition (hERG1a- vs. the b-isoform has recently been reported to alter pharmacologic responses to some hERG blockers and was proposed to be an essential factor pre-disposing patients for drug-induced QT prolongation. Very little is known about the gating and pharmacological properties of two isoforms in heart membranes. For example, how gating mechanisms of the hERG1a channels differ from that of hERG1b is still unknown. The mechanisms by which hERG 1a/1b hetero-tetramers contribute to function in the heart, or what role hERG1b might play in disease are all questions to be answered. Structurally, the two isoforms differ only in the N-terminal region located in the cytoplasm: hERG1b is 340 residues shorter than hERG1a and the initial 36 residues of hERG1b are unique to this isoform. In this study, we combined electrophysiological measurements for HEK cells, kinetics and structural modeling to tease out the individual contributions of each isoform to Action Potential formation and then make predictions about the effects of having various mixture ratios of the two isoforms. By coupling electrophysiological data with computational kinetic modeling, two proposed mechanisms of hERG gating in two homo-tetramers were examined. Sets of data from various experimental stimulation protocols (HEK cells were analyzed simultaneously and fitted to Markov-chain models (M

Isoform-specific regulation of osteogenic factors by polypeptide N-Acetylgalactosaminyltransferases 1 and 4

International Nuclear Information System (INIS)

Tang, Juan; Zheng, Hanxi; Chen, Ling; Gao, Shangshang; Shi, Xiaorui; Liu, Jingjing; Xu, Lan

2017-01-01

The family of UDP-GalNAc polypeptide: N-Acetylgalactosaminlytransfersases (ppGalNAcTs) catalyzes the initial step of O-linked protein glycosylation. Mucin-type O-glycoproteins are abundant in the bone and may play an important role in osteogenesis. Herein, we examined the effects of ppGalNAc-T isoforms on osteogenesis of MC3T3-E1 pre-osteoblasts. We found that ppGalNAc-T1 and -T4 isoforms were highly expressed during osteogenesis of MC3T3-E1 and their knockdown by short hairpin RNA (shRNA) decreased osteoblast formation and bone mineralization. Knockdown of ppGalNAc-T1 or -T4 decreased mRNA and protein levels of bone sialoprotein (BSP). Knockdown of ppGalNAc-T1decreased mRNA levels of osteocalcin (OC), osteoprotegerin (OPG). Knockdown ofppGalNAc-T4 isoform decreased mRNA levels of OC, OPG and vitamin D receptor (VDR). While knockdown of T1 or T4 isoforms did not change the expression of osteopontin (OPN), COLLI, receptor activator for nuclear factor-κB ligand (RANKL) and transforming growth factor-β (TGF-β). Our results demonstrated that the ppGalNAc-T4 was highly expressed in MC3T3-E1 cells during osteogenesis for the first time. We also found that ppGalNAc-T1 and -T4 affected the expression of different osteogenic factors, suggesting distinct roles ppGalNAc-T isoformsplay in regulating osteogenesis in vitro. - Highlights: • ppGalNAc-T1 and T4 are highly expressed during MC3T3 cell osteogenesis. • Knockdown of ppGalNAc-T1 and -T4 decreases osteogenic differentiation and mineralization. • Expression of osteogenic factors are differentially affected by decreased ppGalNAc-T1 and -T4 expression.

Molecular characters and expression analysis of a new isoform of ...

African Journals Online (AJOL)

STORAGESEVER

2008-10-20

Oct 20, 2008 ... isoform of the myocyte enhancer factor 2 gene from the silkworm, Bombyx mori. Qing-zhi Ling1, 2, ... BMEF2B mRNA content in the brain was measured using the combined method of quantitative RT-PCR and Southern ... specific cofactors to control gene expression in pheno- typically different muscles.

Inflammatory Adipokines Decrease Expression of Two High Molecular Weight Isoforms of Tropomyosin Similar to the Change in Type 2 Diabetic Patients.

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Stuart A Savill

Full Text Available Cardiovascular disease and cancer are increased in Type 2 diabetes. TPM1 and TPM4 genes encode proteins associated with cardiovascular and neoplastic disease. High (HMW and low (LMW molecular weight isoforms from TPM1 and TPM4 are altered in several cancer cells and the 3'UTR of TPM1 mRNA is tumour suppressive. Leukocytes influence cardiovascular and neoplastic disease by immunosurveillance for cancer and by chronic inflammation in Type 2 diabetes and cardiovascular disease. The aim was to determine changes in expression of isoforms from TPM1 and TPM4 genes in leukocytes from Type 2 diabetic patients and to use the leukocyte cell line THP1 to identify possible mediators of changes in the patients. Gene expression was determined by RT-qPCR. In diabetes, expression of HMW isoforms from TPM1 were markedly decreased (0.55 v 1.00; p = 0.019 but HMW isoforms from TPM4 were not significantly different (0.76 v 1.00; p = 0.205. Within individual variance in expression of HMW isoforms was very high. The change in expression in HMW isoforms from TPM1 and TPM4 was replicated in THP1 cells treated with 1 ng/ml TNFα (0.10 and 0.12 v 1.00 respectively or 10 ng/ml IL-1α (0.17 and 0.14 v 1.00 respectively. Increased insulin or glucose concentrations had no substantial effects on TPM1 or TPM4 expression. Decreased TPM1 mRNA resulted in decreases in HMW protein levels. Expression of HMW isoforms from TPM1 is decreased in Type 2 diabetes. This is probably due to increased levels of inflammatory cytokines TNFα and IL-1α in Type 2 diabetes. Lower levels of TPM1 mRNA reduce tumour suppression and could contribute to increased cancer risk in Type 2 diabetes. Decreased HMW tropomyosin isoforms are associated with cancer. Decreased HMW isoforms give rise to cells that are more plastic, motile, invasive and prone to dedifferentiation resulting in leukocytes that are more invasive but less functionally effective.

Differential Expression of Sox11 and Bdnf mRNA Isoforms in the Injured and Regenerating Nervous Systems

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Felix L. Struebing

2017-11-01

Full Text Available In both the central nervous system (CNS and the peripheral nervous system (PNS, axonal injury induces changes in neuronal gene expression. In the PNS, a relatively well-characterized alteration in transcriptional activation is known to promote axonal regeneration. This transcriptional cascade includes the neurotrophin Bdnf and the transcription factor Sox11. Although both molecules act to facilitate successful axon regeneration in the PNS, this process does not occur in the CNS. The present study examines the differential expression of Sox11 and Bdnf mRNA isoforms in the PNS and CNS using three experimental paradigms at different time points: (i the acutely injured CNS (retina after optic nerve crush and PNS (dorsal root ganglion after sciatic nerve crush, (ii a CNS regeneration model (retina after optic nerve crush and induced regeneration; and (iii the retina during a chronic form of central neurodegeneration (the DBA/2J glaucoma model. We find an initial increase of Sox11 in both PNS and CNS after injury; however, the expression of Bdnf isoforms is higher in the PNS relative to the CNS. Sustained upregulation of Sox11 is seen in the injured retina following regeneration treatment, while the expression of two Bdnf mRNA isoforms is suppressed. Furthermore, two isoforms of Sox11 with different 3′UTR lengths are present in the retina, and the long isoform is specifically upregulated in later stages of glaucoma. These results provide insight into the molecular cascades active during axonal injury and regeneration in mammalian neurons.

Expression of the human isoform of glutamate dehydrogenase, hGDH2, augments TCA cycle capacity and oxidative metabolism of glutamate during glucose deprivation in astrocytes.

Science.gov (United States)

Nissen, Jakob D; Lykke, Kasper; Bryk, Jaroslaw; Stridh, Malin H; Zaganas, Ioannis; Skytt, Dorte M; Schousboe, Arne; Bak, Lasse K; Enard, Wolfgang; Pääbo, Svante; Waagepetersen, Helle S

2017-03-01

A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO 2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1 and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation and supports the TCA cycle during energy-demanding processes such as high intensity glutamatergic signaling. However, little is known about how expression of hGDH2 affects the handling of glutamate and TCA cycle metabolism in astrocytes. Therefore, we cultured astrocytes from cerebral cortical tissue of hGDH2-expressing transgenic mice. We measured glutamate uptake and metabolism using [ 3 H]glutamate, while the effect on metabolic pathways of glutamate and glucose was evaluated by use of 13 C and 14 C substrates and analysis by mass spectrometry and determination of radioactively labeled metabolites including CO 2 , respectively. We conclude that hGDH2 expression increases capacity for uptake and oxidative metabolism of glutamate, particularly during increased workload and aglycemia. Additionally, hGDH2 expression increased utilization of branched-chain amino acids (BCAA) during aglycemia and caused a general decrease in oxidative glucose metabolism. We speculate, that expression of hGDH2 allows astrocytes to spare glucose and utilize BCAAs during substrate shortages. These findings support the proposed role of hGDH2 in astrocytes as an important fail-safe during situations of intense glutamatergic activity. GLIA 2017;65:474-488. © 2016 Wiley Periodicals, Inc.

Reduced Expression of the Liver/Beta-Cell Glucose Transporter Isoform in Glucose-Insensitive Pancreatic Beta Cells of Diabetic Rats

Science.gov (United States)

Thorens, Bernard; Weir, Gordon C.; Leahy, John L.; Lodish, Harvey F.; Bonner-Weir, Susan

1990-09-01

Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.

Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II

Energy Technology Data Exchange (ETDEWEB)

Boone, Christopher D. [University of Florida, PO Box 100267, Gainesville, FL 32610 (United States); Tu, Chingkuang [University of Florida, PO Box 100245, Gainesville, FL 32610 (United States); McKenna, Robert, E-mail: rmckenna@ufl.edu [University of Florida, PO Box 100267, Gainesville, FL 32610 (United States)

2014-06-01

The structure of human carbonic anhydrase II in complex with cholate has been determined to 1.54 Å resolution. Elucidation of the novel inhibition mechanism of cholate will aid in the development of a nonsulfur-containing, isoform-specific therapeutic agent. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO{sub 2} into bicarbonate and a proton. Human isoform CA II (HCA II) is abundant in the surface epithelial cells of the gastric mucosa, where it serves an important role in cytoprotection through bicarbonate secretion. Physiological inhibition of HCA II via the bile acids contributes to mucosal injury in ulcerogenic conditions. This study details the weak biophysical interactions associated with the binding of a primary bile acid, cholate, to HCA II. The X-ray crystallographic structure determined to 1.54 Å resolution revealed that cholate does not make any direct hydrogen-bond interactions with HCA II, but instead reconfigures the well ordered water network within the active site to promote indirect binding to the enzyme. Structural knowledge of the binding interactions of this nonsulfur-containing inhibitor with HCA II could provide the template design for high-affinity, isoform-specific therapeutic agents for a variety of diseases/pathological states, including cancer, glaucoma, epilepsy and osteoporosis.

Short-term strength training and the expression of myostatin and IGF-I isoforms in rat muscle and tendon

DEFF Research Database (Denmark)

Heinemeier, K M; Olesen, J L; Schjerling, P

2007-01-01

In skeletal muscle, an increased expression of insulin like growth factor-I isoforms IGF-IEa and mechano-growth factor (MGF) combined with downregulation of myostatin is thought to be essential for training-induced hypertrophy. However, the specific effects of different contraction types on regul......In skeletal muscle, an increased expression of insulin like growth factor-I isoforms IGF-IEa and mechano-growth factor (MGF) combined with downregulation of myostatin is thought to be essential for training-induced hypertrophy. However, the specific effects of different contraction types...... on regulation of these factors in muscle are still unclear, and in tendon the functions of myostatin, IGF-IEa, and MGF in relation to training are unknown. Female Sprague-Dawley rats were subjected to 4 days of concentric, eccentric, or isometric training (n = 7-9 per group) of the medial gastrocnemius......, by stimulation of the sciatic nerve during general anesthesia. mRNA levels for myostatin, IGF-IEa, and MGF in muscle and Achilles' tendon were measured by real-time RT-PCR. Muscle myostatin mRNA decreased in response to all types of training (2- to 8-fold) (P

MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori

Science.gov (United States)

Hundreds of Bombyx mori miRNAs had been identified in recent years, but their function in vivo remains poorly understood. The silkworm EcR gene (BmEcR) has three transcriptional isoforms, A, B1 and B2. Isoform sequences are different in the 3’UTR region of the gene, which is the case only in insects...

Peroxiredoxin isoforms are associated with cardiovascular risk factors in type 2 diabetes mellitus

Energy Technology Data Exchange (ETDEWEB)

El Eter, E. [Physiology Department, Faculty of Medicine, King Saud University, Riyadh (Saudi Arabia); Cardiovascular Research Group, Faculty of Medicine, King Saud University, Riyadh (Saudi Arabia); Physiology Department, Faculty of Medicine, Alexandria University, Alexandria (Egypt); Al-Masri, A.A. [Physiology Department, Faculty of Medicine, King Saud University, Riyadh (Saudi Arabia); Cardiovascular Research Group, Faculty of Medicine, King Saud University, Riyadh (Saudi Arabia)

2015-03-03

The production of oxygen free radicals in type 2 diabetes mellitus contributes to the development of complications, especially the cardiovascular-related ones. Peroxiredoxins (PRDXs) are antioxidant enzymes that combat oxidative stress. The aim of this study was to investigate the associations between the levels of PRDX isoforms (1, 2, 4, and 6) and cardiovascular risk factors in type 2 diabetes mellitus. Fifty-three patients with type 2 diabetes mellitus (28F/25M) and 25 healthy control subjects (7F/18M) were enrolled. We measured the plasma levels of each PRDX isoform and analyzed their correlations with cardiovascular risk factors. The plasma PRDX1, -2, -4, and -6 levels were higher in the diabetic patients than in the healthy control subjects. PRDX2 and -6 levels were negatively correlated with diastolic blood pressure, fasting blood sugar, and hemoglobin A1c. In contrast, PRDX1 levels were positively correlated with low-density lipoprotein and C-reactive protein levels. PRDX4 levels were negatively correlated with triglycerides. In conclusion, PRDX1, -2, -4, and -6 showed differential correlations with a variety of traditional cardiovascular risk factors. These results should encourage further research into the crosstalk between PRDX isoforms and cardiovascular risk factors.

Peroxiredoxin isoforms are associated with cardiovascular risk factors in type 2 diabetes mellitus

International Nuclear Information System (INIS)

El Eter, E.; Al-Masri, A.A.

2015-01-01

The production of oxygen free radicals in type 2 diabetes mellitus contributes to the development of complications, especially the cardiovascular-related ones. Peroxiredoxins (PRDXs) are antioxidant enzymes that combat oxidative stress. The aim of this study was to investigate the associations between the levels of PRDX isoforms (1, 2, 4, and 6) and cardiovascular risk factors in type 2 diabetes mellitus. Fifty-three patients with type 2 diabetes mellitus (28F/25M) and 25 healthy control subjects (7F/18M) were enrolled. We measured the plasma levels of each PRDX isoform and analyzed their correlations with cardiovascular risk factors. The plasma PRDX1, -2, -4, and -6 levels were higher in the diabetic patients than in the healthy control subjects. PRDX2 and -6 levels were negatively correlated with diastolic blood pressure, fasting blood sugar, and hemoglobin A1c. In contrast, PRDX1 levels were positively correlated with low-density lipoprotein and C-reactive protein levels. PRDX4 levels were negatively correlated with triglycerides. In conclusion, PRDX1, -2, -4, and -6 showed differential correlations with a variety of traditional cardiovascular risk factors. These results should encourage further research into the crosstalk between PRDX isoforms and cardiovascular risk factors

Amino Acid Profile, Group of Functional and Molecular Weight Distribution of Goat Skin Gelatin That Produced Through Acid Process

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Muhammad Irfan Said

2012-02-01

Full Text Available Gelatin is a product of hydrolysis of collagen protein from animals that are partially processed.  Gelatin used in food and non food industries.  Gelatin is produced when many import of raw skins and bones of pigs and cows.  Goat skins potential as a raw material substitution that still doubt its halal. Process production of gelatin determine the properties of gelatin. The objectives of this research were to determine amino acid profile, group of functional and molecular weight distribution of gelatin made from goat skins which was produced through a process of acid. The skin of male Bligon goat, 1.5 to 2.5 year old was used as raw materials. Process production of gelatin was using acid type acetic acid (CH3COOH 0.5 M (v/v as curing material. The experimental design applied in this study and commercial gelatin was used as control. The results showed that gelatin produced from goat skin through the process of acid had properties identical with commercial gelatin. It can be concluded that the gelatin has the potential substitute product of commercial gelatin. Keywords: collagen, gelatin, goat skin, curing, acid process

Carboxylic acid functional group analysis using constant neutral loss scanning-mass spectrometry

Energy Technology Data Exchange (ETDEWEB)

Dron, Julien [Laboratoire de Chimie et Environnement, Marseille Universites (case 29), 3 place Victor Hugo, 13331 Marseille Cedex 3 (France)], E-mail: julien.dron@up.univ-mrs.fr; Eyglunent, Gregory; Temime-Roussel, Brice; Marchand, Nicolas; Wortham, Henri [Laboratoire de Chimie et Environnement, Marseille Universites (case 29), 3 place Victor Hugo, 13331 Marseille Cedex 3 (France)

2007-12-12

The present study describes the development of a new analytical technique for the functional group determination of the carboxylic moiety using atmospheric pressure chemical ionization-mass spectrometry (APCI-MS/MS) operated in the constant neutral loss scanning (CNLS) mode. Carboxylic groups were first derivatized into their corresponding methyl esters by reacting with BF{sub 3}/methanol mix and the reaction mixture was then directly injected into the APCI chamber. The loss of methanol (m/z = 32 amu) resulting from the fragmentation of the protonated methyl esters was then monitored. Applying this method together with a statistical approach to reference mixtures containing 31 different carboxylic acids at randomly calculated concentrations demonstrated its suitability for quantitative functional group measurements with relative standard deviations below 15% and a detection limit of 0.005 mmol L{sup -1}. Its applicability to environmental matrices was also shown through the determination of carboxylic acid concentrations inside atmospheric aerosol samples. To the best of our knowledge, it is the first time that the tandem mass spectrometry was successfully applied to functional group analysis, offering great perspectives in the characterization of complex mixtures which are prevailing in the field of environmental analysis as well as in the understanding of the chemical processes occurring in these matrices.

Carboxylic acid functional group analysis using constant neutral loss scanning-mass spectrometry

International Nuclear Information System (INIS)

Dron, Julien; Eyglunent, Gregory; Temime-Roussel, Brice; Marchand, Nicolas; Wortham, Henri

2007-01-01

The present study describes the development of a new analytical technique for the functional group determination of the carboxylic moiety using atmospheric pressure chemical ionization-mass spectrometry (APCI-MS/MS) operated in the constant neutral loss scanning (CNLS) mode. Carboxylic groups were first derivatized into their corresponding methyl esters by reacting with BF 3 /methanol mix and the reaction mixture was then directly injected into the APCI chamber. The loss of methanol (m/z = 32 amu) resulting from the fragmentation of the protonated methyl esters was then monitored. Applying this method together with a statistical approach to reference mixtures containing 31 different carboxylic acids at randomly calculated concentrations demonstrated its suitability for quantitative functional group measurements with relative standard deviations below 15% and a detection limit of 0.005 mmol L -1 . Its applicability to environmental matrices was also shown through the determination of carboxylic acid concentrations inside atmospheric aerosol samples. To the best of our knowledge, it is the first time that the tandem mass spectrometry was successfully applied to functional group analysis, offering great perspectives in the characterization of complex mixtures which are prevailing in the field of environmental analysis as well as in the understanding of the chemical processes occurring in these matrices

Hypothyroidism leads to increased collagen-based stiffness and re-expression of large cardiac titin isoforms with high compliance.

Science.gov (United States)

Wu, Yiming; Peng, Jun; Campbell, Kenneth B; Labeit, Siegfried; Granzier, Henk

2007-01-01

Because long-term hypothyroidism results in diastolic dysfunction, we investigated myocardial passive stiffness in hypothyroidism and focused on the possible role of titin, an important determinant of diastolic stiffness. A rat model of hypothyroidism was used, obtained by administering propylthiouracil (PTU) for times that varied from 1 month (short-term) to 4 months (long-term). Titin expression was determined by transcript analysis, gel electrophoresis and immunoelectron microscopy. Diastolic function was measured at the isolated heart, skinned muscle, and cardiac myocyte levels. We found that hypothyroidism resulted in expression of a large titin isoform, the abundance of which gradually increased with time to become the most dominant isoform in long-term hypothyroid rats. This isoform co-migrates on high-resolution gels with fetal cardiac titin. Transcript analysis on myocardium of long-term PTU rats, provided evidence for expression of additional PEVK and Ig domain exons, similar to what has been described in fetal myocardium. Consistent with the expression of a large titin isoform, titin-based restoring and passive forces were significantly reduced in single cardiac myocytes and muscle strips of long-term hypothyroid rats. Overall muscle stiffness and LV diastolic wall stiffness were increased, however, due to increased collagen-based stiffness. We conclude that long term hypothyroidism triggers expression of a large cardiac titin isoform and that the ensuing reduction in titin-based passive stiffness functions as a compensatory mechanism to reduce LV wall stiffness.

Altered β-Amyloid Precursor Protein Isoforms in Mexican Alzheimer’s Disease Patients

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V. J. Sánchez-González

2006-01-01

Full Text Available Objective: To determine the β-amyloid precursor protein (βAPP isoforms ratio as a risk factor for Alzheimer’s Disease and to assess its relationship with demographic and genetic variables of the disease.

A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

Directory of Open Access Journals (Sweden)

Ioannis Gavvovidis

Full Text Available Biallelic mutations in MCPH1 cause primary microcephaly (MCPH with the cellular phenotype of defective chromosome condensation. MCPH1 encodes a multifunctional protein that notably is involved in brain development, regulation of chromosome condensation, and DNA damage response. In the present studies, we detected that MCPH1 encodes several distinct transcripts, including two major forms: full-length MCPH1 (MCPH1-FL and a second transcript lacking the six 3' exons (MCPH1Δe9-14. Both variants show comparable tissue-specific expression patterns, demonstrate nuclear localization that is mediated independently via separate NLS motifs, and are more abundant in certain fetal than adult organs. In addition, the expression of either isoform complements the chromosome condensation defect found in genetically MCPH1-deficient or MCPH1 siRNA-depleted cells, demonstrating a redundancy of both MCPH1 isoforms for the regulation of chromosome condensation. Strikingly however, both transcripts are regulated antagonistically during cell-cycle progression and there are functional differences between the isoforms with regard to the DNA damage response; MCPH1-FL localizes to phosphorylated H2AX repair foci following ionizing irradiation, while MCPH1Δe9-14 was evenly distributed in the nucleus. In summary, our results demonstrate here that MCPH1 encodes different isoforms that are differentially regulated at the transcript level and have different functions at the protein level.

A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

Science.gov (United States)

Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

2013-11-13

Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

Characterization of acid functional groups of carbon dots by nonlinear regression data fitting of potentiometric titration curves

Science.gov (United States)

Alves, Larissa A.; de Castro, Arthur H.; de Mendonça, Fernanda G.; de Mesquita, João P.

2016-05-01

The oxygenated functional groups present on the surface of carbon dots with an average size of 2.7 ± 0.5 nm were characterized by a variety of techniques. In particular, we discussed the fit data of potentiometric titration curves using a nonlinear regression method based on the Levenberg-Marquardt algorithm. The results obtained by statistical treatment of the titration curve data showed that the best fit was obtained considering the presence of five Brønsted-Lowry acids on the surface of the carbon dots with constant ionization characteristics of carboxylic acids, cyclic ester, phenolic and pyrone-like groups. The total number of oxygenated acid groups obtained was 5 mmol g-1, with approximately 65% (∼2.9 mmol g-1) originating from groups with pKa titrated and initial concentration of HCl solution. Finally, we believe that the methodology used here, together with other characterization techniques, is a simple, fast and powerful tool to characterize the complex acid-base properties of these so interesting and intriguing nanoparticles.

Alteration of protein expression pattern of vascular endothelial growth factor (VEGF) from soluble to cell-associated isoform during tumourigenesis

International Nuclear Information System (INIS)

Cressey, Ratchada; Wattananupong, Onusa; Lertprasertsuke, Nirush; Vinitketkumnuen, Usanee

2005-01-01

colorectal and 1,251 ± 568 pg/ml in lung) than a healthy volunteer group (543 ± 344 pg/ml). No correlation between the level of circulating VEGF and the pathologic features of tumours was observed. Our findings indicate that the expression patterns of VEGF isoforms are altered during tumourigenesis as certain isoform overexpression in tumour tissues correlated with tumour progression indicating their important role in tumour development. However, measurement of VEGF in the circulation as a prognostic marker needs to be carefully evaluated as the cell-associated isoform (VEGF 189 ), but not the soluble isoform (VEGF 121 and VEGF 165 ) appears to play important role in tumour progression

Acidity enhancement of unsaturated bases of group 15 by association with borane and beryllium dihydride. Unexpected boron and beryllium Brønsted acids.

Science.gov (United States)

Martín-Sómer, Ana; Mó, Otilia; Yáñez, Manuel; Guillemin, Jean-Claude

2015-01-21

The intrinsic acidity of CH2[double bond, length as m-dash]CHXH2, HC[triple bond, length as m-dash]CXH2 (X = N, P, As, Sb) derivatives and of their complexes with BeH2 and BH3 has been investigated by means of high-level density functional theory and molecular orbital ab initio calculations, using as a reference the ethyl saturated analogues. The acidity of the free systems steadily increases down the group for the three series of derivatives, ethyl, vinyl and ethynyl. The association with both beryllium dihydride and borane leads to a very significant acidity enhancement, being larger for BeH2 than for BH3 complexes. This acidity enhancement, for the unsaturated compounds, is accompanied by a change in the acidity trends down the group, which do not steadily decrease but present a minimum value for both the vinyl- and the ethynyl-phosphine. When the molecule acting as the Lewis acid is beryllium dihydride, the π-type complexes in which the BeH2 molecules interact with the double or triple bond are found, in some cases, to be more stable, in terms of free energies, than the conventional complexes in which the attachment takes place at the heteroatom, X. The most important finding, however, is that P, As, and Sb ethynyl complexes with BeH2 do not behave as P, As, or Sb Brønsted acids, but unexpectedly as Be acids.

Isoform-Selective Disruption of AKAP-Localized PKA Using Hydrocarbon Stapled Peptides

Science.gov (United States)

2015-01-01

A-kinase anchoring proteins (AKAPs) play an important role in the spatial and temporal regulation of protein kinase A (PKA) by scaffolding critical intracellular signaling complexes. Here we report the design of conformationally constrained peptides that disrupt interactions between PKA and AKAPs in an isoform-selective manner. Peptides derived from the A Kinase Binding (AKB) domain of several AKAPs were chemically modified to contain an all-hydrocarbon staple and target the docking/dimerization domain of PKA-R, thereby occluding AKAP interactions. The peptides are cell-permeable against diverse human cell lines, are highly isoform-selective for PKA-RII, and can effectively inhibit interactions between AKAPs and PKA-RII in intact cells. These peptides can be applied as useful reagents in cell-based studies to selectively disrupt AKAP-localized PKA-RII activity and block AKAP signaling complexes. In summary, the novel hydrocarbon-stapled peptides developed in this study represent a new class of AKAP disruptors to study compartmentalized RII-regulated PKA signaling in cells. PMID:24422448

High-throughput proteomics detection of novel splice isoforms in human platelets.

LENUS (Irish Health Repository)

Power, Karen A

2009-01-01

Alternative splicing (AS) is an intrinsic regulatory mechanism of all metazoans. Recent findings suggest that 100% of multiexonic human genes give rise to splice isoforms. AS can be specific to tissue type, environment or developmentally regulated. Splice variants have also been implicated in various diseases including cancer. Detection of these variants will enhance our understanding of the complexity of the human genome and provide disease-specific and prognostic biomarkers. We adopted a proteomics approach to identify exon skip events - the most common form of AS. We constructed a database harboring the peptide sequences derived from all hypothetical exon skip junctions in the human genome. Searching tandem mass spectrometry (MS\\/MS) data against the database allows the detection of exon skip events, directly at the protein level. Here we describe the application of this approach to human platelets, including the mRNA-based verification of novel splice isoforms of ITGA2, NPEPPS and FH. This methodology is applicable to all new or existing MS\\/MS datasets.

Axodendritic sorting and pathological missorting of Tau are isoform-specific and determined by axon initial segment architecture.

Science.gov (United States)

Zempel, Hans; Dennissen, Frank J A; Kumar, Yatender; Luedtke, Julia; Biernat, Jacek; Mandelkow, Eva-Maria; Mandelkow, Eckhard

2017-07-21

Subcellular mislocalization of the microtubule-associated protein Tau is a hallmark of Alzheimer disease (AD) and other tauopathies. Six Tau isoforms, differentiated by the presence or absence of a second repeat or of N-terminal inserts, exist in the human CNS, but their physiological and pathological differences have long remained elusive. Here, we investigated the properties and distributions of human and rodent Tau isoforms in primary forebrain rodent neurons. We found that the Tau diffusion barrier (TDB), located within the axon initial segment (AIS), controls retrograde (axon-to-soma) and anterograde (soma-to-axon) traffic of Tau. Tau isoforms without the N-terminal inserts were sorted efficiently into the axon. However, the longest isoform (2N4R-Tau) was partially retained in cell bodies and dendrites, where it accelerated spine and dendrite growth. The TDB (located within the AIS) was impaired when AIS components (ankyrin G, EB1) were knocked down or when glycogen synthase kinase-3β (GSK3β; an AD-associated kinase tethered to the AIS) was overexpressed. Using superresolution nanoscopy and live-cell imaging, we observed that microtubules within the AIS appeared highly dynamic, a feature essential for the TDB. Pathomechanistically, amyloid-β insult caused cofilin activation and F-actin remodeling and decreased microtubule dynamics in the AIS. Concomitantly with these amyloid-β-induced disruptions, the AIS/TDB sorting function failed, causing AD-like Tau missorting. In summary, we provide evidence that the human and rodent Tau isoforms differ in axodendritic sorting and amyloid-β-induced missorting and that the axodendritic distribution of Tau depends on AIS integrity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Managing brain extracellular K+ during neuronal activity: The physiological role of the Na+/K+-ATPase subunit isoforms

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Brian Roland eLarsen

2016-04-01

Full Text Available AbstractDuring neuronal activity in the brain, extracellular K+ rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K+ is the Na+/K+-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na+/K+-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K+ from neurons, whereas the neurons themselves become the primary K+ absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na+/K+-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K+ from the extracellular space in the face of neuronal activity.Understanding the nature, impact and effects of the various Na+/K+-ATPase isoform combinations in K+ management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2 and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood. This review addresses aspects of the Na+/K+-ATPase in the regulation of extracellular K+ in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease.

Simultaneous Detection of Human C-Terminal p53 Isoforms by Single Template Molecularly Imprinted Polymers (MIPs) Coupled with Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)-Based Targeted Proteomics.

Science.gov (United States)

Jiang, Wenting; Liu, Liang; Chen, Yun

2018-03-06

Abnormal expression of C-terminal p53 isoforms α, β, and γ can cause the development of cancers including breast cancer. To date, much evidence has demonstrated that these isoforms can differentially regulate target genes and modulate their expression. Thus, quantification of individual isoforms may help to link clinical outcome to p53 status and to improve cancer patient treatment. However, there are few studies on accurate determination of p53 isoforms, probably due to sequence homology of these isoforms and also their low abundance. In this study, a targeted proteomics assay combining molecularly imprinted polymers (MIPs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for simultaneous quantification of C-terminal p53 isoforms. Isoform-specific surrogate peptides (i.e., KPLDGEYFTLQIR (peptide-α) for isoform α, KPLDGEYFTLQDQTSFQK (peptide-β) for isoform β, and KPLDGEYFTLQMLLDLR (peptide-γ) for isoform γ) were first selected and used in both MIPs enrichment and mass spectrometric detection. The common sequence KPLDGEYFTLQ of these three surrogate peptides was used as single template in MIPs. In addition to optimization of imprinting conditions and characterization of the prepared MIPs, binding affinity and cross-reactivity of the MIPs for each surrogate peptide were also evaluated. As a result, a LOQ of 5 nM was achieved, which was >15-fold more sensitive than that without MIPs. Finally, the assay was validated and applied to simultaneous quantitative analysis of C-terminal p53 isoforms α, β, and γ in several human breast cell lines (i.e., MCF-10A normal cells, MCF-7 and MDA-MB-231 cancer cells, and drug-resistant MCF-7/ADR cancer cells). This study is among the first to employ single template MIPs and cross-reactivity phenomenon to select isoform-specific surrogate peptides and enable simultaneous quantification of protein isoforms in LC-MS/MS-based targeted proteomics.

Isoforms of thyroxine-binding globulin as a model for molecular epidemiology of human cancer risk

International Nuclear Information System (INIS)

Golovaty, A.S.; Lapko, A.G.

2000-01-01

-binding globulin (TBG) by 50-60%, but no elevation in free T4, as compared with similar control groups of from Minsk. One of the reasons for the euthyroid syndrome in teenagers from the contaminated region was shown to be the blood isoforms in TBG. These isoforms differed in the structure of the carbohydrate components and as a rule are characterized by a prolonged circulation time. Using specific affinity chromatography, with subsequent immunological assay we shown that a certain amount of serum TBG-molecules in persons from Khojniki contains a high level of TBG (about 30% higher than the control group) containing fucosyalated biantennary sugar chains with more prolonged survival time. Previous findings showed that increased levels of fucosylation are observed for some serum glycoproteins in the blood of tumour patients. We provide evidence for variations in the TBGfuc which are contained in the serum TBG pool of patients suffering from cancer with various localisation. Thus, analysis of TBGfuc may be regarded as a prognostic marker in the determination of risk groups. The molecular mechanism of increased activity of fucosyl transferases will be discussed. We propose to measure the level of TBG fucosylated to assess the human cancer risk and habitants of contaminated region of Belarus. (authors)

Expression of glutamic acid decarboxylase and identification of GABAergic cells in the ischemic rat dentate gyrus

DEFF Research Database (Denmark)

Müller, Georg Johannes; Dogonowski, Anne-Marie; Finsen, Bente

2006-01-01

We have investigated the glutamic acid dexcarboxylase (GAD) mRNA and protein isoforms as markers for ischemic loss of GABAergic neurons in the dentate hilus. Stereological counts of these neurons were performed in rats surviving 8 days after 10 min of transient forebrain ischemia, and in control...

Roles of SGK Isoform Signaling in Breast Cancer Migration and Invasion

Science.gov (United States)

2011-04-01

significant number of overlapping substrates, and deregulation in breast carcinoma (5,6). To date, no studies have investigated any role for SGK in cell...isoforms in breast carcinoma cell lines (months 2-3) To insure specificity of SGK knockdown in breast cancer cell lines I made two different specific

IgE recognition of chimeric isoforms of the honeybee (Apis mellifera) venom allergen Api m 10 evaluated by protein array technology.

Science.gov (United States)

Van Vaerenbergh, Matthias; De Smet, Lina; Rafei-Shamsabadi, David; Blank, Simon; Spillner, Edzard; Ebo, Didier G; Devreese, Bart; Jakob, Thilo; de Graaf, Dirk C

2015-02-01

Api m 10 has recently been established as novel major allergen that is recognized by more than 60% of honeybee venom (HBV) allergic patients. Previous studies suggest Api m 10 protein heterogeneity which may have implications for diagnosis and immunotherapy of HBV allergy. In the present study, RT-PCR revealed the expression of at least nine additional Api m 10 transcript isoforms by the venom glands. Two distinct mechanisms are responsible for the generation of these isoforms: while the previously known variant 2 is produced by an alternative splicing event, novel identified isoforms are intragenic chimeric transcripts. To the best of our knowledge, this is the first report of the identification of chimeric transcripts generated by the honeybee. By a retrospective proteomic analysis we found evidence for the presence of several of these isoforms in the venom proteome. Additionally, we analyzed IgE reactivity to different isoforms by protein array technology using sera from HBV allergic patients, which revealed that IgE recognition of Api m 10 is both isoform- and patient-specific. While it was previously demonstrated that the majority of HBV allergic patients display IgE reactivity to variant 2, our study also shows that some patients lacking IgE antibodies for variant 2 display IgE reactivity to two of the novel identified Api m 10 variants, i.e. variants 3 and 4. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterisation of Cdkl5transcript isoforms in rat

OpenAIRE

Hector, Ralph D.; Dando, Owen; Ritakari, Tuula E.; Kind, Peter C.; Bailey, Mark E.S.; Cobb, Stuart R.

2017-01-01

CDKL5 deficiency is a severe neurological disorder caused by mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5). The predominant human CDKL5 brain isoform is a 9.7kb transcript comprised of 18 exons with a large 6.6kb 3'-untranslated region (UTR). Mammalian models of CDKL5 disorder are currently limited to mouse, and little is known about Cdkl5 in other organisms used to model neurodevelopmental disorders, such as rat. In this study we characterise, both bioinformatically a...

Oxidation of an activated carbon commercial and characterization of the content of superficial acid groups

International Nuclear Information System (INIS)

Cortes, Juan Carlos; Giraldo Liliana; Garcia, Andres A; Garcia, Cesar; Moreno, Juan C

2008-01-01

The changes of the surface acid groups of an activated commercial carbon after placing it under oxidation treatment with nitric acid are studied. The time used was in the range 1.5 and 9 hours, the concentrations range was from 4 to 7 molL -1 . The study included the determination of immersion enthalpy. Boehm's type titrations, FTIR, and pH at the point of zero charge, pH p zc. It was found that total acid groups are in a range from 0.207 mmolg -1 to 1.247 mmolg -1 , and that they are proportional to the immersion enthalpy in NaOH that are between 40 and 54Jg -1 . The pH p zc decreases with the oxidation treatment and have values between 8.3 and 4.3

Preparation of New Adsorbent Containing Hydroxamic Acid Groups by Electron Beam-Induced Grafting for Metal Ion Adsorption

International Nuclear Information System (INIS)

Suwanmala, Phiriyatorn; Hoshina, Hiroyuki; Seko, Noriaki; Tamada, Masao

2007-08-01

Full text: A new adsorbent containing hydroxamic acid groups was synthesized by electron beam-induced graft copolymerization of methyl acrylate (MA) onto nonwoven fabric composed of polyethylene-coated polypropylene fiber. Conversion of ester groups of the grafted copolymer into the hydroxamic groups was performed by treatment with an alkaline solution of hydroxylamine (HA). Adsorbent containing hydroxamic acid groups can adsorb 99% of UO2 2+ , 98% of V5+, 97% of Pb2+ and 96% of Al3+ at pH, 5, 4, 6, and 4, respectively, after coming into contact with 100 ppb metal solution for 24 h

Quadrupole time-of-flight mass spectometry : a method to study the actual expression of allergen isoforms identified by PCR cloning

NARCIS (Netherlands)

Helsper, J.P.F.G.; Gilissen, L.J.W.J.; Ree, van R.; America, A.H.P.; Cordewener, J.H.G.; Bosch, D.

2002-01-01

Background: Over the past 2 decades, molecular biology has shown that most major allergens exist in multiple isoforms. Very little is known about the relevance of allergen isoforms at the level of expressed protein (ie, actual allergen exposure). Objective: The aim of this study was to evaluate the

Quadrupole time-of-flight mass spectrometry: a method to study the actual expression of allergen isoforms identified by PCR cloning

NARCIS (Netherlands)

Helsper, Johannes P. F. G.; Gilissen, Luud J. W. J.; van Ree, Ronald; America, Antoine H. P.; Cordewener, Jan H. G.; Bosch, Dirk

2002-01-01

BACKGROUND: Over the past 2 decades, molecular biology has shown that most major allergens exist in multiple isoforms. Very little is known about the relevance of allergen isoforms at the level of expressed protein (ie, actual allergen exposure). OBJECTIVE: The aim of this study was to evaluate the

Role of L-type Ca2+ channel isoforms in the extinction of conditioned fear.

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Busquet, Perrine; Hetzenauer, Alfred; Sinnegger-Brauns, Martina J; Striessnig, Jörg; Singewald, Nicolas

2008-05-01

Dihydropyridine (DHP) L-type Ca(2+) channel (LTCC) antagonists, such as nifedipine, have been reported to impair the extinction of conditioned fear without interfering with its acquisition. Identification of the LTCC isoforms mediating this DHP effect is an essential basis to reveal their role as potential drug targets for the treatment of specific anxiety disorders. Ca(V)1.2 and Ca(V)1.3 are the predominant LTCCs in the mammalian brain. However, since no isoform-selective DHP blockers are available, their individual contribution to fear memory extinction is unknown. We used a novel mouse model expressing DHP-insensitive Ca(V)1.2 LTCCs (Ca(V)1.2DHP(-/-) mice) to address this question. In line with previous studies, wild-type (WT) mice treated with systemic nifedipine displayed markedly impaired fear extinction. This DHP effect was completely abolished in Ca(V)1.2DHP(-/-) mice, indicating that it is mediated by Ca(V)1.2, but not by Ca(V)1.3 LTCCs. Supporting this conclusion, Ca(V)1.3-deficient mice (Ca(V)1.3(-/-)) showed extinction identical to the respective WT mice. The inhibition of fear extinction was not observed after intracerebroventricular (i.c.v.) application of different doses of nifedipine, suggesting that this effect is secondary to inhibition of peripheral Ca(V)1.2 channels. The LTCC activator BayK, which lacks neurotoxic effects in Ca(V)1.2DHP(-/-) mice, did not influence the extinction time course. In summary, we demonstrate that LTCC signaling through the Ca(V)1.2 isoform of LTCCs interferes with fear memory extinction, presumably via a peripherally mediated mechanism. Activation of other LTCC isoforms (predominantly Ca(V)1.3) is not sufficient to accelerate extinction of conditioned fear in mice.

Inhibition of PaCaMKII-E isoform in the dorsal unpaired median neurosecretory cells of cockroach reduces nicotine- and clothianidin-induced currents.

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List, Olivier; Calas-List, Delphine; Taillebois, Emiliane; Juchaux, Marjorie; Heuland, Emilie; Thany, Steeve H

2014-08-01

Cellular responses to Ca(2+) require intermediary proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII), which transduces the signal into downstream effects. We recently demonstrated that the cockroach genome encodes five different CaMKII isoforms, and only PaCaMKII-E isoform is specifically expressed in the dorsal unpaired median neurosecretory cells. In the present study, using antisense oligonucleotides, we demonstrated that PaCaMKII-E isoform inhibition reduced nicotine-induced currents through α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptor subtypes. Specifically, PaCaMKII-E isoform is sufficient to repress nicotinic current amplitudes as a result of its depression by antisense oligonucleotides. Similar results were found using the neonicotinoid insecticide clothianidin, which acted as a full agonist of dorsal unpaired median neuron nicotinic acetylcholine receptors. Clothianidin current amplitudes are strongly reduced under bath application of PaCaMKII-E antisense oligonucleotides but no significant results are found with α-bungarotoxin co-applied, demonstrating that CaMKII-E isoform affects nicotine currents through α-bungarotoxin-sensitive and -insensitive receptor subtypes whereas clothianidin currents are reduced via α-bungarotoxin-insensitive receptors. In addition, we found that intracellular calcium increase induced by nicotine and clothianidin were reduced by PaCaMKII-E antisense oligonucleotides, demonstrating that intracellular calcium increase induced by nicotine and clothianidin are affected by PaCaMKII-E inhibition. Cellular responses to Ca(2+) require intermediary proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII). We recently demonstrated that the cockroach genome encodes five different CaMKII isoforms and only PaCaMKII-E isoform was specifically expressed in the dorsal unpaired median neurosecretory cells. Here we show that specific inhibition of PaCaMKII-E isoform is

Conditional expression of CD44 isoforms in lymphoma cells: influence on hyaluronate binding and tumor growth

Energy Technology Data Exchange (ETDEWEB)

Fu, J.

2002-03-01

CD44 describes a family of surface proteins consisting of many isoforms due to alternative splice of ten 'variant' exons. Members of this family are involved in various processes including hematopoiesis, lymphocyte activation and homing, limb development, wound healing and tumor progression. Clinically, CD44 has been shown to be a prognostic factor for several human cancers. To answer the question which isoform might be relevant for tumor progression and to gain an insight into the mechanism of its function, I established transfectants of the LB lymphoma cell line in which the expression of four CD44 isoforms, namely CD44v3-10, CD44v4-10, CD44v8-10 and CD44s, was controlled by the Tet-off promoter. In the presence of Doxycycline, the expression was repressed. Removal of Doxycycline switched on expression and the maximal CD44 amount was obtained within two days. The transfectants were characterized regarding their ability to bind to the extracellular matrix component hyaluronate (HA). Overexpression of all four CD44 isoforms conferred the ability to bind HA on LB cells. Other glycosaminoglycans (GAGs) were bound in an isotype-specific fashion. CD44v3-10, CD44v4-10 and CD44v8-10 showed high binding affinity to chondroitin A, B and C, and low affinity to heparin, heparan sulfate and keratan sulfate. CD44s could not bind to these GAGs. Among these three variants, the binding ability of CD44v3-10 was the strongest. CD44 clustering seemed to play a crucial role for HA binding. Both CD44s and CD44v8-10 formed reduction-sensitive complexes in LB cells. The complexes are homooligomers or heterooligomers composed of different isoforms. Cys286 in CD44 transmember domain was not responsible for the formation of reduction-sensitive oligomer or for the enhanced HA binding in LB cell line. Using a conditional dimerization system the requirement of CD44 oligomerization for HA binding was directly demonstrated. The induction of oligomerization increased HA binding

Conditional expression of CD44 isoforms in lymphoma cells: influence on hyaluronate binding and tumor growth

International Nuclear Information System (INIS)

Fu, J.

2002-03-01

CD44 describes a family of surface proteins consisting of many isoforms due to alternative splice of ten 'variant' exons. Members of this family are involved in various processes including hematopoiesis, lymphocyte activation and homing, limb development, wound healing and tumor progression. Clinically, CD44 has been shown to be a prognostic factor for several human cancers. To answer the question which isoform might be relevant for tumor progression and to gain an insight into the mechanism of its function, I established transfectants of the LB lymphoma cell line in which the expression of four CD44 isoforms, namely CD44v3-10, CD44v4-10, CD44v8-10 and CD44s, was controlled by the Tet-off promoter. In the presence of Doxycycline, the expression was repressed. Removal of Doxycycline switched on expression and the maximal CD44 amount was obtained within two days. The transfectants were characterized regarding their ability to bind to the extracellular matrix component hyaluronate (HA). Overexpression of all four CD44 isoforms conferred the ability to bind HA on LB cells. Other glycosaminoglycans (GAGs) were bound in an isotype-specific fashion. CD44v3-10, CD44v4-10 and CD44v8-10 showed high binding affinity to chondroitin A, B and C, and low affinity to heparin, heparan sulfate and keratan sulfate. CD44s could not bind to these GAGs. Among these three variants, the binding ability of CD44v3-10 was the strongest. CD44 clustering seemed to play a crucial role for HA binding. Both CD44s and CD44v8-10 formed reduction-sensitive complexes in LB cells. The complexes are homooligomers or heterooligomers composed of different isoforms. Cys286 in CD44 transmember domain was not responsible for the formation of reduction-sensitive oligomer or for the enhanced HA binding in LB cell line. Using a conditional dimerization system the requirement of CD44 oligomerization for HA binding was directly demonstrated. The induction of oligomerization increased HA binding. Finally, I

Two Isoforms of Yersinia pestis Plasminogen Activator Pla: Intraspecies Distribution, Intrinsic Disorder Propensity, and Contribution to Virulence.

Science.gov (United States)

Dentovskaya, Svetlana V; Platonov, Mikhail E; Svetoch, Tat'yana E; Kopylov, Pavel Kh; Kombarova, Tat'yana I; Ivanov, Sergey A; Shaikhutdinova, Rima Z; Kolombet, Lyubov' V; Chauhan, Sadhana; Ablamunits, Vitaly G; Motin, Vladimir L; Uversky, Vladimir N; Anisimov, Andrey P

2016-01-01

It has been shown previously that several endemic Y. pestis isolates with limited virulence contained the I259 isoform of the outer membrane protease Pla, while the epidemic highly virulent strains possessed only the T259 Pla isoform. Our sequence analysis of the pla gene from 118 Y. pestis subsp. microtus strains revealed that the I259 isoform was present exclusively in the endemic strains providing a convictive evidence of more ancestral origin of this isoform. Analysis of the effects of the I259T polymorphism on the intrinsic disorder propensity of Pla revealed that the I259T mutation slightly increases the intrinsic disorder propensity of the C-terminal tail of Pla and makes this protein slightly more prone for disorder-based protein-protein interactions, suggesting that the T259 Pla could be functionally more active than the I259 Pla. This assumption was proven experimentally by assessing the coagulase and fibrinolytic activities of the two Pla isoforms in human plasma, as well as in a direct fluorometric assay with the Pla peptide substrate. The virulence testing of Pla-negative or expressing the I259 and T259 Pla isoforms Y. pestis subsp. microtus and subsp. pestis strains did not reveal any significant difference in LD50 values and dose-dependent survival assays between them by using a subcutaneous route of challenge of mice and guinea pigs or intradermal challenge of mice. However, a significant decrease in time-to-death was observed in animals infected with the epidemic T259 Pla-producing strains as compared to the parent Pla-negative variants. Survival curves of the endemic I259 Pla+ strains fit between them, but significant difference in mean time to death post infection between the Pla-strains and their I259 Pla+ variants could be seen only in the isogenic set of subsp. pestis strains. These findings suggest an essential role for the outer membrane protease Pla evolution in Y. pestis bubonic infection exacerbation that is necessary for intensification

Temporal, Diagnostic, and Tissue-Specific Regulation of NRG3 Isoform Expression in Human Brain Development and Affective Disorders

Science.gov (United States)

Paterson, Clare; Wang, Yanhong; Hyde, Thomas M.; Weinberger, Daniel R.; Kleinman, Joel E.; Law, Amanda J.

2018-01-01

Objective Genes implicated in schizophrenia are enriched in networks differentially regulated during human CNS development. Neuregulin 3 (NRG3), a brain-enriched neurotrophin, undergoes alternative splicing and is implicated in several neurological disorders with developmental origins. Isoform-specific increases in NRG3 are observed in schizophrenia and associated with rs10748842, a NRG3 risk polymorphism, suggesting NRG3 transcriptional dysregulation as a molecular mechanism of risk. The authors quantitatively mapped the temporal trajectories of NRG3 isoforms (classes I–IV) in the neocortex throughout the human lifespan, examined whether tissue-specific regulation of NRG3 occurs in humans, and determined if abnormalities in NRG3 transcriptomics occur in mood disorders and are genetically determined. Method NRG3 isoform classes I–IV were quantified using quantitative real-time polymerase chain reaction in human postmortem dorsolateral prefrontal cortex from 286 nonpsychiatric control individuals, from gestational week 14 to 85 years old, and individuals diagnosed with either bipolar disorder (N=34) or major depressive disorder (N=69). Tissue-specific mapping was investigated in several human tissues. rs10748842 was genotyped in individuals with mood disorders, and association with NRG3 isoform expression examined. Results NRG3 classes displayed individually specific expression trajectories across human neocortical development and aging; classes I, II, and IV were significantly associated with developmental stage. NRG3 class I was increased in bipolar and major depressive disorder, consistent with observations in schizophrenia. NRG3 class II was increased in bipolar disorder, and class III was increased in major depression. The rs10748842 risk genotype predicted elevated class II and III expression, consistent with previous reports in the brain, with tissue-specific analyses suggesting that classes II and III are brain-specific isoforms of NRG3. Conclusions

Profound human/mouse differences in alpha-dystrobrevin isoforms: a novel syntrophin-binding site and promoter missing in mouse and rat

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Jin Hong

2009-12-01

Full Text Available Abstract Background The dystrophin glycoprotein complex is disrupted in Duchenne muscular dystrophy and many other neuromuscular diseases. The principal heterodimeric partner of dystrophin at the heart of the dystrophin glycoprotein complex in the main clinically affected tissues (skeletal muscle, heart and brain is its distant relative, α-dystrobrevin. The α-dystrobrevin gene is subject to complex transcriptional and post-transcriptional regulation, generating a substantial range of isoforms by alternative promoter use, alternative polyadenylation and alternative splicing. The choice of isoform is understood, amongst other things, to determine the stoichiometry of syntrophins (and their ligands in the dystrophin glycoprotein complex. Results We show here that, contrary to the literature, most α-dystrobrevin genes, including that of humans, encode three distinct syntrophin-binding sites, rather than two, resulting in a greatly enhanced isoform repertoire. We compare in detail the quantitative tissue-specific expression pattern of human and mouse α-dystrobrevin isoforms, and show that two major gene features (the novel syntrophin-binding site-encoding exon and the internal promoter and first exon of brain-specific isoforms α-dystrobrevin-4 and -5 are present in most mammals but specifically ablated in mouse and rat. Conclusion Lineage-specific mutations in the murids mean that the mouse brain has fewer than half of the α-dystrobrevin isoforms found in the human brain. Our finding that there are likely to be fundamental functional differences between the α-dystrobrevins (and therefore the dystrophin glycoprotein complexes of mice and humans raises questions about the current use of the mouse as the principal model animal for studying Duchenne muscular dystrophy and other related disorders, especially the neurological aspects thereof.

Alternatively spliced CD44 isoforms containing exon v10 promote cellular adhesion through the recognition of chondroitin sulfate-modified CD44

NARCIS (Netherlands)

Chiu, R K; Droll, A; Dougherty, S T; Carpenito, C; Cooper, D L; Dougherty, G J

1999-01-01

Correlations have been noted between the expression of certain alternatively spliced CD44 isoforms and the metastatic propensity of various histologically distinct tumor cell types. The precise mechanism by which particular CD44 isoforms contribute to the metastatic process is, however, unclear. In

Expression Profile of Cationic Amino Acid Transporters in Rats with Endotoxin-Induced Uveitis

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Yung-Ray Hsu

2016-01-01

Full Text Available Purpose. The transcellular arginine transportation via cationic amino acid transporter (CAT is the rate-limiting step in nitric oxide (NO synthesis, which is crucial in intraocular inflammation. In this study, CAT isoforms and inducible nitric oxide synthase (iNOS expression was investigated in endotoxin-induced uveitis (EIU. Methods. EIU was induced in Lewis rats by lipopolysaccharide (LPS injection. In the treatment group, the rats were injected intraperitoneally with the proteasome inhibitor bortezomib before EIU induction. After 24 hours, leukocyte quantification, NO measurement of the aqueous humor, and histopathological examination were evaluated. The expression of CAT isoforms and iNOS was determined by reverse transcription-polymerase chain reaction, western blotting, and immunofluorescence staining. Nuclear factor-kappa B (NF-κB binding activity was evaluated by electrophoretic mobility shift assay. The mouse macrophage cell line RAW 264.7 was used to validate the in vivo findings. Results. LPS significantly stimulated iNOS, CAT-2A, and CAT-2B mRNA and protein expression but did not affect CAT-1 in EIU rats and RAW 264.7 cells. Bortezomib attenuated inflammation and inhibited iNOS, CAT-2A, and CAT-2B expression through NF-κB inhibition. Conclusions. CAT-2 and iNOS, but not CAT-1, are specifically involved in EIU. NF-κB is essential in the induction of CAT-2 and iNOS in EIU.

p110α and p110β isoforms of PI3K signaling: are they two sides of the same coin?

Science.gov (United States)

Singh, Paramjeet; Dar, Mohd Saleem; Dar, Mohd Jamal

2016-09-01

Class-1 phosphatidylinositol-3-kinases (PI3Ks) are activated by a variety of extracellular stimuli and have been implicated in a wide range of cellular processes. p110α and p110β are the two most studied isoforms of the class-1A PI3K signaling pathway. Although these two isoforms are ubiquitously expressed and play multiple redundant roles, they also have distinct functions within the cell. More recently, p110α and p110β isoforms have been shown to translocate into the nucleus and play a role in DNA replication and repair, and in cell cycle progression. In the following Review article, we discuss the overlapping and unique roles of p110α and p110β isoforms with a particular focus on their structure, expression analysis, subcellular localization, and signaling contributions in various cell types and model organisms. © 2016 Federation of European Biochemical Societies.

The pokeweed leaf mRNA transcriptome and its regulation by jasmonic acid.

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Kira C.M. Neller

2016-03-01

Full Text Available The American pokeweed plant, Phytolacca americana, is recognized for synthesizing pokeweed antiviral protein (PAP, a ribosome inactivating protein (RIP that inhibits the replication of several plant and animal viruses. The plant is also a heavy metal accumulator with applications in soil remediation. However, little is known about pokeweed stress responses, as large-scale sequencing projects have not been performed for this species. Here, we sequenced the mRNA transcriptome of pokeweed in the presence and absence of jasmonic acid (JA, a hormone mediating plant defense. Trinity-based de novo assembly of mRNA from leaf tissue and BLASTx homology searches against public sequence databases resulted in the annotation of 59 096 transcripts. Differential expression analysis identified JA-responsive genes that may be involved in defense against pathogen infection and herbivory. We confirmed the existence of several PAP isoforms and cloned a potentially novel isoform of PAP. Expression analysis indicated that PAP isoforms are differentially responsive to JA, perhaps indicating specialized roles within the plant. Finally, we identified 52 305 natural antisense transcript pairs, four of which comprised PAP isoforms, suggesting a novel form of RIP gene regulation. This transcriptome-wide study of a Phytolaccaceae family member provides a source of new genes that may be involved in stress tolerance in this plant. The sequences generated in our study have been deposited in the SRA database under project # SRP069141.

A Review of Metallothionein Isoforms and their Role in Pathophysiology

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Senthil kumar M

2011-05-01

Full Text Available Abstract The Metallothionein (MT is a protein which has several interesting biological effects and has been demonstrated increase focus on the role of MT in various biological systems in the past three decades. The studies on the role of MT were limited with few areas like apoptosis and antioxidants in selected organs even fifty years after its discovery. Now acknowledge the exploration of various isoforms of MT such as MT-I, MT-II, MT-III and MT-IV and other isoforms in various biological systems. Strong evidence exists that MT modulates complex diseases and the immune system in the body but the primary function of MT still remains unknown. This review's main objective is to explore the capability to specifically manipulate MT levels in cells and in animals to provide answers regarding how MT could impact those complex disease scenarios. The experimental result mentioned in this review related among MT, zinc, cadmium, diabetic, heart disease, bone retardation, neuro toxicity, kidney dysfunction, cancer, and brain suggest novel method for exploration and contribute significantly to the growing scientist to research further in this field.

Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP.

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Helena Hernández

2009-09-01

Full Text Available Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B'. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.

Non-Muscle Myosin II Isoforms Have Different Functions in Matrix Rearrangement by MDA-MB-231 Cells.

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Bridget Hindman

Full Text Available The role of a stiffening extra-cellular matrix (ECM in cancer progression is documented but poorly understood. Here we use a conditioning protocol to test the role of nonmuscle myosin II isoforms in cell mediated ECM arrangement using collagen constructs seeded with breast cancer cells expressing shRNA targeted to either the IIA or IIB heavy chain isoform. While there are several methods available to measure changes in the biophysical characteristics of the ECM, we wanted to use a method which allows for the measurement of global stiffness changes as well as a dynamic response from the sample over time. The conditioning protocol used allows the direct measurement of ECM stiffness. Using various treatments, it is possible to determine the contribution of various construct and cellular components to the overall construct stiffness. Using this assay, we show that both the IIA and IIB isoforms are necessary for efficient matrix remodeling by MDA-MB-231 breast cancer cells, as loss of either isoform changes the stiffness of the collagen constructs as measured using our conditioning protocol. Constructs containing only collagen had an elastic modulus of 0.40 Pascals (Pa, parental MDA-MB-231 constructs had an elastic modulus of 9.22 Pa, while IIA and IIB KD constructs had moduli of 3.42 and 7.20 Pa, respectively. We also calculated the cell and matrix contributions to the overall sample elastic modulus. Loss of either myosin isoform resulted in decreased cell stiffness, as well as a decrease in the stiffness of the cell-altered collagen matrices. While the total construct modulus for the IIB KD cells was lower than that of the parental cells, the IIB KD cell-altered matrices actually had a higher elastic modulus than the parental cell-altered matrices (4.73 versus 4.38 Pa. These results indicate that the IIA and IIB heavy chains play distinct and non-redundant roles in matrix remodeling.

Synaptic dysbindin-1 reductions in schizophrenia occur in an isoform-specific manner indicating their subsynaptic location.

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Konrad Talbot

Full Text Available BACKGROUND: An increasing number of studies report associations between variation in DTNBP1, a top candidate gene in schizophrenia, and both the clinical symptoms of the disorder and its cognitive deficits. DTNBP1 encodes dysbindin-1, reduced levels of which have been found in synaptic fields of schizophrenia cases. This study determined whether such synaptic reductions are isoform-specific. METHODOLOGY/PRINCIPAL FINDINGS: Using Western blotting of tissue fractions, we first determined the synaptic localization of the three major dysbindin-1 isoforms (A, B, and C. All three were concentrated in synaptosomes of multiple brain areas, including auditory association cortices in the posterior half of the superior temporal gyrus (pSTG and the hippocampal formation (HF. Tests on the subsynaptic tissue fractions revealed that each isoform is predominantly, if not exclusively, associated with synaptic vesicles (dysbindin-1B or with postsynaptic densities (dysbindin-1A and -1C. Using Western blotting on pSTG (n = 15 and HF (n = 15 synaptosomal fractions from schizophrenia cases and their matched controls, we discovered that synaptic dysbindin-1 is reduced in an isoform-specific manner in schizophrenia without changes in levels of synaptophysin or PSD-95. In pSTG, about 92% of the schizophrenia cases displayed synaptic dysbindin-1A reductions averaging 48% (p = 0.0007 without alterations in other dysbindin-1 isoforms. In the HF, by contrast, schizophrenia cases displayed normal levels of synaptic dysbindin-1A, but 67% showed synaptic reductions in dysbindin-1B averaging 33% (p = 0.0256, while 80% showed synaptic reductions in dysbindin-1C averaging 35% (p = 0.0171. CONCLUSIONS/SIGNIFICANCE: Given the distinctive subsynaptic localization of dysbindin-1A, -1B, and -1C across brain regions, the observed pSTG reductions in dysbindin-1A are postsynaptic and may promote dendritic spine loss with consequent disruption of auditory information

Study of amino acid disorders among a high risk group of Egyptian ...

African Journals Online (AJOL)

Aim of the work: The present work aimed at investigating infants (In neonatal and post neonatal period) and children suspected of having inborn errors of metabolism with unexplained mental retardation. The frequency pattern of the various amino acid disorders, in a group of selected infants and children was done to ...

Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

Science.gov (United States)

Ocampo, J.; McCormack, B.; Navarro, E.; Moreno, S.; Garre, V.

2012-01-01

The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. PMID:22635921

Amino acids analysis using grouping and parceling of neutrons cross sections techniques

International Nuclear Information System (INIS)

Voi, Dante Luiz Voi; Rocha, Helio Fenandes da

2002-01-01

Amino acids used in parenteral administration in hospital patients with special importance in nutritional applications were analyzed to compare with the manufactory data. Individual amino acid samples of phenylalanine, cysteine, methionine, tyrosine and threonine were measured with the neutron crystal spectrometer installed at the J-9 irradiation channel of the 1 kW Argonaut Reactor of the Instituto de Engenharia Nuclear (IEN). Gold and D 2 O high purity samples were used for the experimental system calibration. Neutron cross section values were calculated from chemical composition, conformation and molecular structure analysis of the materials. Literature data were manipulated by parceling and grouping neutron cross sections. (author)

Constitutive and nitrogen catabolite repression-sensitive production of Gat1 isoforms.

Science.gov (United States)

Rai, Rajendra; Tate, Jennifer J; Georis, Isabelle; Dubois, Evelyne; Cooper, Terrance G

2014-01-31

Nitrogen catabolite repression (NCR)-sensitive transcription is activated by Gln3 and Gat1. In nitrogen excess, Gln3 and Gat1 are cytoplasmic, and transcription is minimal. In poor nitrogen, Gln3 and Gat1 become nuclear and activate transcription. A long standing paradox has surrounded Gat1 production. Gat1 was first reported as an NCR-regulated activity mediating NCR-sensitive transcription in gln3 deletion strains. Upon cloning, GAT1 transcription was, as predicted, NCR-sensitive and Gln3- and Gat1-activated. In contrast, Western blots of Gat1-Myc(13) exhibited two constitutively produced species. Investigating this paradox, we demonstrate that wild type Gat1 isoforms (IsoA and IsoB) are initiated at Gat1 methionines 40, 95, and/or 102, but not at methionine 1. Their low level production is the same in rich and poor nitrogen conditions. When the Myc(13) tag is placed after Gat1 Ser-233, four N-terminal Gat1 isoforms (IsoC-F) are also initiated at methionines 40, 95, and/or 102. However, their production is highly NCR-sensitive, being greater in proline than glutamine medium. Surprisingly, all Gat1 isoforms produced in sufficient quantities to be confidently analyzed (IsoA, IsoC, and IsoD) require Gln3 and UASGATA promoter elements, both requirements typical of NCR-sensitive transcription. These data demonstrate that regulated Gat1 production is more complex than previously recognized, with wild type versus truncated Gat1 proteins failing to be regulated in parallel. This is the first reported instance of Gln3 UASGATA-dependent protein production failing to derepress in nitrogen poor conditions. A Gat1-lacZ ORF swap experiment indicated sequence(s) responsible for the nonparallel production are downstream of Gat1 leucine 61.

The Allosterically Unregulated Isoform of ADP-Glucose Pyrophosphorylase from Barley Endosperm Is the Most Likely Source of ADP-Glucose Incorporated into Endosperm Starch.

Science.gov (United States)

Doan; Rudi; Olsen

1999-11-01

We present the results of studies of an unmodified version of the recombinant major barley (Hordeum vulgare) endosperm ADP-glucose pyrophoshorylase (AGPase) expressed in insect cells, which corroborate previous data that this isoform of the enzyme acts independently of the allosteric regulators 3-phosphoglycerate and inorganic phosphate. We also present a characterization of the individual subunits expressed separately in insect cells, showing that the SS AGPase is active in the presence of 3-phosphoglycerate and is inhibited by inorganic phosphate. As a step toward the elucidation of the role of the two AGPase isoforms in barley, the temporal and spatial expression profile of the four barley AGPase transcripts encoding these isoforms were studied. The results show that the steady-state level of beps and bepl, the transcripts encoding the major endosperm isoform, correlated positively with the rate of endosperm starch accumulation. In contrast, blps and blpl, the transcripts encoding the major leaf isoform, were constitutively expressed at a very low steady-state level throughout the barley plant. The implications of these findings for the evolution of plant AGPases are discussed.

Implementation of a SPR immunosensor for the simultaneous detection of the 22K and 20K hGH isoforms in human serum samples.

Science.gov (United States)

de Juan-Franco, Elena; Rodríguez-Frade, J M; Mellado, M; Lechuga, Laura M

2013-09-30

We have implemented a Surface Plasmon Resonance (SPR) immunosensor based on a sandwich assay for the simultaneous detection of the two main hGH isoforms, of 22 kDa (22K) and 20 kDa (20K). An oriented-antibody sensor surface specific for both hormone isoforms was assembled by using the biotin-streptavidin system. The immunosensor functionality was checked for the direct detection of the 22K hGH isoform in buffer, which gave high specificity and reproducibility (intra and inter-assay mean coefficients of variation of 8.23% and 9% respectively). The selective determination of the 22K and 20K hGH isoforms in human serum samples in a single assay was possible by using two specific anti-hGH monoclonal antibodies. The detection limit for both hormone isoforms was 0.9 ng mL(-1) and the mean coefficient of variation was below 7.2%. The excellent reproducibility and sensitivity obtained indicate the high performance of this immunosensor for implementing an anti-doping test. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular Analysis of Collagen XVIII Reveals Novel Mutations, Presence of a Third Isoform, and Possible Genetic Heterogeneity in Knobloch Syndrome

Science.gov (United States)

Suzuki, O. T.; Sertié, A. L.; Der Kaloustian, V. M.; Kok, F.; Carpenter, M.; Murray, J.; Czeizel, A. E.; Kliemann, S. E.; Rosemberg, S.; Monteiro, M.; Olsen, B. R.; Passos-Bueno, M. R.

2002-01-01

Knobloch syndrome (KS) is a rare disease characterized by severe ocular alterations, including vitreoretinal degeneration associated with retinal detachment and occipital scalp defect. The responsible gene, COL18A1, has been mapped to 21q22.3, and, on the basis of the analysis of one family, we have demonstrated that a mutation affecting only one of the three COL18A1 isoforms causes this phenotype. We report here the results of the screening of both the entire coding region and the exon-intron boundaries of the COL18A1 gene (which includes 43 exons), in eight unrelated patients with KS. Besides 20 polymorphic changes, we identified 6 different pathogenic changes in both alleles of five unrelated patients with KS (three compound heterozygotes and two homozygotes). All are truncating mutations leading to deficiency of one or all collagen XVIII isoforms and endostatin. We have verified that, in exon 41, the deletion c3514-3515delCT, found in three unrelated alleles, is embedded in different haplotypes, suggesting that this mutation has occurred more than once. In addition, our results provide evidence of nonallelic genetic heterogeneity in KS. We also show that the longest human isoform (NC11-728) is expressed in several tissues (including the human eye) and that lack of either the short variant or all of the collagen XVIII isoforms causes similar phenotypes but that those patients who lack all forms present more-severe ocular alterations. Despite the small sample size, we found low endostatin plasma levels in those patients with mutations leading to deficiency of all isoforms; in addition, it seems that absence of all collagen XVIII isoforms causes predisposition to epilepsy. PMID:12415512

Differential expression of hERG1 channel isoforms reproduces properties of native I(Kr and modulates cardiac action potential characteristics.

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Anders Peter Larsen

Full Text Available BACKGROUND: The repolarizing cardiac rapid delayed rectifier current, I(Kr, is composed of ERG1 channels. It has been suggested that two isoforms of the ERG1 protein, ERG1a and ERG1b, both contribute to I(Kr. Marked heterogeneity in the kinetic properties of native I(Kr has been described. We hypothesized that the heterogeneity of native I(Kr can be reproduced by differential expression of ERG1a and ERG1b isoforms. Furthermore, the functional consequences of differential expression of ERG1 isoforms were explored as a potential mechanism underlying native heterogeneity of action potential duration (APD and restitution. METHODOLOGY/PRINCIPAL FINDINGS: The results show that the heterogeneity of native I(Kr can be reproduced in heterologous expression systems by differential expression of ERG1a and ERG1b isoforms. Characterization of the macroscopic kinetics of ERG1 currents demonstrated that these were dependent on the relative abundance of ERG1a and ERG1b. Furthermore, we used a computational model of the ventricular cardiomyocyte to show that both APD and the slope of the restitution curve may be modulated by varying the relative abundance of ERG1a and ERG1b. As the relative abundance of ERG1b was increased, APD was gradually shortened and the slope of the restitution curve was decreased. CONCLUSIONS/SIGNIFICANCE: Our results show that differential expression of ERG1 isoforms may explain regional heterogeneity of I(Kr kinetics. The data demonstrate that subunit dependent changes in channel kinetics are important for the functional properties of ERG1 currents and hence I(Kr. Importantly, our results suggest that regional differences in the relative abundance of ERG1 isoforms may represent a potential mechanism underlying the heterogeneity of both APD and APD restitution observed in mammalian hearts.

Selective antagonists at group I metabotropic glutamate receptors: synthesis and molecular pharmacology of 4-aryl-3-isoxazolol amino acids

DEFF Research Database (Denmark)

Kromann, Hasse; Sløk, Frank A; Stensbøl, Tine B

2002-01-01

Homologation of (S)-glutamic acid (Glu, 1) and Glu analogues has previously provided ligands with activity at metabotropic Glu receptors (mGluRs). The homologue of ibotenic acid (7), 2-amino-3-(3-hydroxy-5-isoxazolyl)propionic acid (HIBO, 8), and the 4-phenyl derivative of 8, compound 9a, are bot...... antagonists at group I mGluRs. Here we report the synthesis and molecular pharmacology of HIBO analogues 9b-h containing different 4-aryl substituents. All of these compounds possess antagonist activity at group I mGluRs but are inactive at group II and III mGluRs....

Acid-Group-Content-Dependent Proton Conductivity Mechanisms at the Interlayer of Poly(N-dodecylacrylamide-co-acrylic acid) Copolymer Multilayer Nanosheet Films.

Science.gov (United States)

Sato, Takuma; Tsukamoto, Mayu; Yamamoto, Shunsuke; Mitsuishi, Masaya; Miyashita, Tokuji; Nagano, Shusaku; Matsui, Jun

2017-11-14

The effect of the content of acid groups on the proton conductivity at the interlayer of polymer-nanosheet assemblies was investigated. For that purpose, amphiphilic poly(N-dodecylacrylamide-co-acrylic acid) copolymers [p(DDA/AA)] with varying contents of AA were synthesized by free radical polymerization. Surface pressure (π)-area (A) isotherms of these copolymers indicated that stable polymer monolayers are formed at the air/water interface for AA mole fraction (n) ≤ 0.49. In all cases, a uniform dispersion of the AA groups in the polymer monolayer was observed. Subsequently, polymer monolayers were transferred onto solid substrates using the Langmuir-Blodgett (LB) technique. X-ray diffraction (XRD) analyses of the multilayer films showed strong Bragg diffraction peaks, suggesting a highly uniform lamellar structure for the multilayer films. The proton conductivity of the multilayer films parallel to the direction of the layer planes were measured by impedance spectroscopy, which revealed that the conductivity increased with increasing values of n. Activation energies for proton conduction of ∼0.3 and 0.42 eV were observed for n ≥ 0.32 and n = 0.07, respectively. Interestingly, the proton conductivity of a multilayer film with n = 0.19 did not follow the Arrhenius equation. These results were interpreted in terms of the average distance between the AA groups (l AA ), and it was concluded that, for n ≥ 0.32, an advanced 2D hydrogen bonding network was formed, while for n = 0.07, l AA is too long to form such hydrogen bonding networks. The l AA for n = 0.19 is intermediate to these extremes, resulting in the formation of hydrogen bonding networks at low temperatures, and disruption of these networks at high temperatures due to thermally induced motion. These results indicate that a high proton conductivity with low activation energy can be achieved, even under weakly acidic conditions, by arranging the acid groups at an optimal distance.

Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat.

Science.gov (United States)

Arakaki, Xianghong; McCleary, Paige; Techy, Matthew; Chiang, Jiarong; Kuo, Linus; Fonteh, Alfred N; Armstrong, Brian; Levy, Dan; Harrington, Michael G

2013-03-14

Cerebrospinal fluid (CSF) sodium concentration increases during migraine attacks, and both CSF and vitreous humor sodium increase in the rat migraine model. The Na,K-ATPase is a probable source of these sodium fluxes. Since Na,K-ATPase isoforms have different locations and physiological roles, our objective was to establish which alpha isoforms are present at sites where sodium homeostasis is disrupted. Specific Na,K-ATPase alpha isoforms were identified in rat tissues by immunohistochemistry at the blood-CSF barrier at the choroid plexus, at the blood-CSF-trigeminal barrier at the meninges, at the blood-retina barrier, and at the blood-aqueous barrier at the ciliary body. Calcitonin gene-related peptide (CGRP), occludin, or von Willibrand factor (vWF) were co-localized with Na,K-ATPase to identify trigeminal nociceptor fibers, tight junctions, and capillary endothelial cells respectively. The Na,K-ATPase alpha-2 isoform is located on capillaries and intensely at nociceptive trigeminal nerve fibers at the meningeal blood-CSF-trigeminal barrier. Alpha-1 and -3 are lightly expressed on the trigeminal nerve fibers but not at capillaries. Alpha-2 is expressed at the blood-retina barriers and, with alpha-1, at the ciliary body blood aqueous barrier. Intense apical membrane alpha-1 was associated with moderate cytoplasmic alpha-2 expression at the choroid plexus blood-CSF barrier. Na,K-ATPase alpha isoforms are present at the meningeal, choroid plexus, and retinal barriers. Alpha-2 predominates at the capillary endothelial cells in the meninges and retinal ganglion cell layer.

Analysis of a lin-42/period Null Allele Implicates All Three Isoforms in Regulation of Caenorhabditis elegans Molting and Developmental Timing

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Theresa L. B. Edelman

2016-12-01

Full Text Available The Caenorhabditis elegans heterochronic gene pathway regulates the relative timing of events during postembryonic development. lin-42, the worm homolog of the circadian clock gene, period, is a critical element of this pathway. lin-42 function has been defined by a set of hypomorphic alleles that cause precocious phenotypes, in which later developmental events, such as the terminal differentiation of hypodermal cells, occur too early. A subset of alleles also reveals a significant role for lin-42 in molting; larval stages are lengthened and ecdysis often fails in these mutant animals. lin-42 is a complex locus, encoding overlapping and nonoverlapping isoforms. Although existing alleles that affect subsets of isoforms have illuminated important and distinct roles for this gene in developmental timing, molting, and the decision to enter the alternative dauer state, it is essential to have a null allele to understand all of the roles of lin-42 and its individual isoforms. To remedy this problem and discover the null phenotype, we engineered an allele that deletes the entire lin-42 protein-coding region. lin-42 null mutants are homozygously viable, but have more severe phenotypes than observed in previously characterized hypomorphic alleles. We also provide additional evidence for this conclusion by using the null allele as a base for reintroducing different isoforms, showing that each isoform can provide heterochronic and molting pathway activities. Transcript levels of the nonoverlapping isoforms appear to be under coordinate temporal regulation, despite being driven by independent promoters. The lin-42 null allele will continue to be an important tool for dissecting the functions of lin-42 in molting and developmental timing.

alpha isoforms of soluble and membrane-linked folate-binding protein in human blood

DEFF Research Database (Denmark)

Hoier-Madsen, M.; Holm, J.; Hansen, S.I.

2008-01-01

supported the hypothesis that serum FBP (29 kDa) mainly originates from neutrophils. The presence of FBP/FR alpha isoforms were established for the first time in human blood using antibodies specifically directed against human milk FBP alpha. The alpha isoforms identified on erythrocyte membranes......, and in granulocytes and serum, only constituted an almost undetectable fraction of the functional FBP The FBP alpha in neutrophil granulocytes was identified as a cytoplasmic component by indirect immunofluorescence. Gel filtration of serum revealed a peak of FBP alpha (>120 kDa), which could represent receptor...... fragments from decomposed erythrocytes and granulocytes. The soluble FBPs may exert bacteriostatic effects and protect folates in plasma from biological degradation, whereas FRs on the surface of blood cells could be involved in intracellular folate uptake or serve as signal proteins. The latter receptors...

Protein kinase C isoforms at the neuromuscular junction: localization and specific roles in neurotransmission and development.

Science.gov (United States)

Lanuza, Maria A; Santafe, Manel M; Garcia, Neus; Besalduch, Núria; Tomàs, Marta; Obis, Teresa; Priego, Mercedes; Nelson, Phillip G; Tomàs, Josep

2014-01-01

The protein kinase C family (PKC) regulates a variety of neural functions including neurotransmitter release. The selective activation of a wide range of PKC isoforms in different cells and domains is likely to contribute to the functional diversity of PKC phosphorylating activity. In this review, we describe the isoform localization, phosphorylation function, regulation and signalling of the PKC family at the neuromuscular junction. Data show the involvement of the PKC family in several important functions at the neuromuscular junction and in particular in the maturation of the synapse and the modulation of neurotransmission in the adult. © 2013 Anatomical Society.

Monoamine Oxidase Inhibitory Activity of Ferulic Acid Amides: Curcumin-Based Design and Synthesis.

Science.gov (United States)

Badavath, Vishnu N; Baysal, İpek; Uçar, Gülberk; Mondal, Susanta K; Sinha, Barij N; Jayaprakash, Venkatesan

2016-01-01

Ferulic acid has structural similarity with curcumin which is being reported for its monoamine oxidase (MAO) inhibitory activity. Based on this similarity, we designed a series of ferulic acid amides 6a-m and tested for their inhibitory activity on human MAO (hMAO) isoforms. All the compounds were found to inhibit the hMAO isoforms either selectively or non-selectively. Nine compounds (6a, 6b, 6g-m) were found to inhibit hMAO-B selectively, whereas the other four (6c-f) were found to be non-selective. There is a gradual shift from hMAO-B selectivity (6a,b) to non-selectivity (6c-f) as there is an increase in chain length at the amino terminus. In case of compounds having an aromatic nucleus at the amino terminus, increasing the carbon number between N and the aromatic ring increases the potency as well as selectivity toward hMAO-B. Compounds 6f, 6j, and 6k were subjected to membrane permeability and metabolic stability studies by in vitro assay methods. They were found to have a better pharmacokinetic profile than curcumin, ferulic acid, and selegiline. In order to understand the structural features responsible for the potency and selectivity of 6k, we carried out a molecular docking simulation study. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

Science.gov (United States)

Hatton, Jason P.; Gaubert, Francois; Cazenave, Jean-Pierre; Schmitt, Didier; Hashemi, B. B. (Principal Investigator); Hughes-Fulford, M. (Principal Investigator)

2002-01-01

Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

Phosphoric acid doped polysulfone membranes with aminopyridine pendant groups and imidazole cross-links

DEFF Research Database (Denmark)

Hink, Steffen; Elsøe, Katrine; Cleemann, Lars Nilausen

2015-01-01

Udel polysulfone based membranes with 4-aminopyridine pendant groups and cross-linking imidazole units are synthesized in a simple two step reaction. The ratio of 4-aminopyridine and imidazole is varied and the materials are extensively characterized. The average phosphoric acid uptake (in 85 wt%...

The Allosterically Unregulated Isoform of ADP-Glucose Pyrophosphorylase from Barley Endosperm Is the Most Likely Source of ADP-Glucose Incorporated into Endosperm Starch1

Science.gov (United States)

Doan, Danny N.P.; Rudi, Heidi; Olsen, Odd-Arne

1999-01-01

We present the results of studies of an unmodified version of the recombinant major barley (Hordeum vulgare) endosperm ADP-glucose pyrophoshorylase (AGPase) expressed in insect cells, which corroborate previous data that this isoform of the enzyme acts independently of the allosteric regulators 3-phosphoglycerate and inorganic phosphate. We also present a characterization of the individual subunits expressed separately in insect cells, showing that the SS AGPase is active in the presence of 3-phosphoglycerate and is inhibited by inorganic phosphate. As a step toward the elucidation of the role of the two AGPase isoforms in barley, the temporal and spatial expression profile of the four barley AGPase transcripts encoding these isoforms were studied. The results show that the steady-state level of beps and bepl, the transcripts encoding the major endosperm isoform, correlated positively with the rate of endosperm starch accumulation. In contrast, blps and blpl, the transcripts encoding the major leaf isoform, were constitutively expressed at a very low steady-state level throughout the barley plant. The implications of these findings for the evolution of plant AGPases are discussed. PMID:10557246

Oestrogen receptor beta isoform expression in sporadic colorectal cancer, familial adenomatous polyposis and progressive stages of colorectal cancer.

Science.gov (United States)

Stevanato Filho, Paulo Roberto; Aguiar Júnior, Samuel; Begnami, Maria Dirlei; Kuasne, Hellen; Spencer, Ranyell Matheus; Nakagawa, Wilson Toshihiko; Bezerra, Tiago Santoro; Kupper, Bruna Catin; Takahashi, Renata Maymi; Barros Filho, Mateus; Rogatto, Silvia Regina; Lopes, Ademar