WorldWideScience

Sample records for synthase protein levels

  1. Carotenoid crystal formation in Arabidopsis and carrot roots caused by increased phytoene synthase protein levels.

    Directory of Open Access Journals (Sweden)

    Dirk Maass

    Full Text Available BACKGROUND: As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism. METHODOLOGY/PRINCIPAL FINDINGS: In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 microg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to beta-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals. CONCLUSIONS: The sequestration of carotenoids into crystals can be driven by the

  2. Locally Different Endothelial Nitric Oxide Synthase Protein Levels in Ascending Aortic Aneurysms of Bicuspid and Tricuspid Aortic Valve

    Directory of Open Access Journals (Sweden)

    Salah A. Mohamed

    2012-01-01

    Full Text Available Aims. Dysregulated expression of the endothelial nitric oxide synthase (eNOS is observed in aortic aneurysms associated with bicuspid aortic valve (BAV. We determined eNOS protein levels in various areas in ascending aortic aneurysms. Methods and Results. Aneurysmal specimens were collected from 19 patients, 14 with BAV and 5 with tricuspid aortic valve (TAV. ENOS protein levels were measured in the outer curve (convexity, the opposite side (concavity, the distal and above the sinotubular junction (proximal aneurysm. Cultured aortic cells were treated with NO synthesis inhibitor L-NAME and the amounts of 35 apoptosis-related proteins were determined. In patients with BAV, eNOS levels were significantly lower in the proximal aorta than in the concavity and distal aorta. ENOS protein levels were also lower in the convexity than in the concavity. While the convexity and distal aorta showed similar eNOS protein levels in BAV and TAV patients, levels were higher in TAV proximal aorta. Inhibition of NO synthesis in aneurysmal aortic cells by L-NAME led to a cytosolic increase in the levels of mitochondrial serine protease HTRA2/Omi. Conclusion. ENOS protein levels were varied at different areas of the aneurysmal aorta. The dysregulation of nitric oxide can lead to an increase in proapoptotic HTRA2/Omi.

  3. Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

    Directory of Open Access Journals (Sweden)

    Louw Abraham I

    2010-04-01

    Full Text Available Abstract Background Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn were examined on a morphological, transcriptomic, proteomic and metabolic level. Results Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in

  4. Thymidylate synthase protein expression levels remain stable during paclitaxel and carboplatin treatment in non-small cell lung cancer

    DEFF Research Database (Denmark)

    Jakobsen, Jan Nyrop; Santoni-Rugiu, Eric; Sørensen, Jens Benn

    2014-01-01

    BACKGROUND: Thymidylate synthase (TS) is a potential predictive marker for efficacy of treatment with pemetrexed. The current study aimed at investigating whether TS expression changes during non-pemetrexed chemotherapy of non-small cell lung cancer (NSCLC), thus making rebiopsy necessary for dec...

  5. The cellulose synthase companion proteins act non-redundantly with CELLULOSE SYNTHASE INTERACTING1/POM2 and CELLULOSE SYNTHASE 6

    OpenAIRE

    Endler, Anne; Schneider, Rene; Kesten, Christopher; Lampugnani, Edwin R.; Persson, Staffan

    2016-01-01

    Cellulose is a cell wall constituent that is essential for plant growth and development, and an important raw material for a range of industrial applications. Cellulose is synthesized at the plasma membrane by massive cellulose synthase (CesA) complexes that track along cortical microtubules in elongating cells of Arabidopsis through the activity of the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1). In a recent study we identified another family of proteins that also are associated with the ...

  6. Sphingomyelin synthases regulate protein trafficking and secretion.

    Directory of Open Access Journals (Sweden)

    Marimuthu Subathra

    Full Text Available Sphingomyelin synthases (SMS1 and 2 represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG. SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein, protein kinase D (PKD, to the Golgi. Since PKD recruitment to the Golgi has been implicated in cellular secretion through the trans golgi network (TGN, the effect of down-regulation of SMSs on TGN-to-plasma membrane trafficking was studied. Down regulation of either SMS1 or SMS2 significantly retarded trafficking of the reporter protein vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP from the TGN to the cell surface. Inhibition of SMSs also induced tubular protrusions from the trans Golgi network reminiscent of inhibited TGN membrane fission. Since a recent study demonstrated the requirement of PKD activity for insulin secretion in beta cells, we tested the function of SMS in this model. Inhibition of SMS significantly reduced insulin secretion in rat INS-1 cells. Taken together these results provide the first direct evidence that both enzymes (SMS1 and 2 are capable of regulating TGN-mediated protein trafficking and secretion, functions that are compatible with PKD being a down-stream target for SMSs in the Golgi.

  7. Glycogen synthase and phosphofructokinase protein and mRNA levels in skeletal muscle from insulin-resistant patients with non-insulin-dependent diabetes mellitus

    DEFF Research Database (Denmark)

    Vestergaard, H; Lund, S; Larsen, F S

    1993-01-01

    In patients with non-insulin-dependent diabetes mellitus (NIDDM) and matched control subjects we examined the interrelationships between in vivo nonoxidative glucose metabolism and glucose oxidation and the muscle activities, as well as the immunoreactive protein and mRNA levels of the rate-limit...

  8. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    Science.gov (United States)

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. The evolution of function in strictosidine synthase-like proteins.

    Science.gov (United States)

    Hicks, Michael A; Barber, Alan E; Giddings, Lesley-Ann; Caldwell, Jenna; O'Connor, Sarah E; Babbitt, Patricia C

    2011-11-01

    The exponential growth of sequence data provides abundant information for the discovery of new enzyme reactions. Correctly annotating the functions of highly diverse proteins can be difficult, however, hindering use of this information. Global analysis of large superfamilies of related proteins is a powerful strategy for understanding the evolution of reactions by identifying catalytic commonalities and differences in reaction and substrate specificity, even when only a few members have been biochemically or structurally characterized. A comparison of >2500 sequences sharing the six-bladed β-propeller fold establishes sequence, structural, and functional links among the three subgroups of the functionally diverse N6P superfamily: the arylesterase-like and senescence marker protein-30/gluconolactonase/luciferin-regenerating enzyme-like (SGL) subgroups, representing enzymes that catalyze lactonase and related hydrolytic reactions, and the so-called strictosidine synthase-like (SSL) subgroup. Metal-coordinating residues were identified as broadly conserved in the active sites of all three subgroups except for a few proteins from the SSL subgroup, which have been experimentally determined to catalyze the quite different strictosidine synthase (SS) reaction, a metal-independent condensation reaction. Despite these differences, comparison of conserved catalytic features of the arylesterase-like and SGL enzymes with the SSs identified similar structural and mechanistic attributes between the hydrolytic reactions catalyzed by the former and the condensation reaction catalyzed by SS. The results also suggest that despite their annotations, the great majority of these >500 SSL sequences do not catalyze the SS reaction; rather, they likely catalyze hydrolytic reactions typical of the other two subgroups instead. This prediction was confirmed experimentally for one of these proteins. Copyright © 2011 Wiley-Liss, Inc.

  10. SIRT3 deacetylates ATP synthase F1 complex proteins in response to nutrient- and exercise-induced stress.

    Science.gov (United States)

    Vassilopoulos, Athanassios; Pennington, J Daniel; Andresson, Thorkell; Rees, David M; Bosley, Allen D; Fearnley, Ian M; Ham, Amy; Flynn, Charles Robb; Hill, Salisha; Rose, Kristie Lindsey; Kim, Hyun-Seok; Deng, Chu-Xia; Walker, John E; Gius, David

    2014-08-01

    Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis. By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCP(K139) directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype. This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins. Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins.

  11. Sesquiterpene Synthase-3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Fusion Protein Responsible for Hirsutene Biosynthesis in Stereum hirsutum.

    Science.gov (United States)

    Flynn, Christopher M; Schmidt-Dannert, Claudia

    2018-06-01

    The wood-rotting mushroom Stereum hirsutum is a known producer of a large number of namesake hirsutenoids, many with important bioactivities. Hirsutenoids form a structurally diverse and distinct class of sesquiterpenoids. No genes involved in hirsutenoid biosynthesis have yet been identified or their enzymes characterized. Here, we describe the cloning and functional characterization of a hirsutene synthase as an unexpected fusion protein of a sesquiterpene synthase (STS) with a C-terminal 3-hydroxy-3-methylglutaryl-coenzyme A (3-hydroxy-3-methylglutaryl-CoA) synthase (HMGS) domain. Both the full-length fusion protein and truncated STS domain are highly product-specific 1,11-cyclizing STS enzymes with kinetic properties typical of STSs. Complementation studies in Saccharomyces cerevisiae confirmed that the HMGS domain is also functional in vivo Phylogenetic analysis shows that the hirsutene synthase domain does not form a clade with other previously characterized sesquiterpene synthases from Basidiomycota. Comparative gene structure analysis of this hirsutene synthase with characterized fungal enzymes reveals a significantly higher intron density, suggesting that this enzyme may be acquired by horizontal gene transfer. In contrast, the HMGS domain is clearly related to other fungal homologs. This STS-HMGS fusion protein is part of a biosynthetic gene cluster that includes P450s and oxidases that are expressed and could be cloned from cDNA. Finally, this unusual fusion of a terpene synthase to an HMGS domain, which is not generally recognized as a key regulatory enzyme of the mevalonate isoprenoid precursor pathway, led to the identification of additional HMGS duplications in many fungal genomes, including the localization of HMGSs in other predicted sesquiterpenoid biosynthetic gene clusters. IMPORTANCE Hirsutenoids represent a structurally diverse class of bioactive sesquiterpenoids isolated from fungi. Identification of their biosynthetic pathways will provide

  12. Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2.

    Directory of Open Access Journals (Sweden)

    Angela Lavado-Roldán

    2016-07-01

    Full Text Available One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2 acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation.

  13. Protein modelling of triterpene synthase genes from mangrove plants using Phyre2 and Swiss-model

    Science.gov (United States)

    Basyuni, M.; Wati, R.; Sulistiyono, N.; Hayati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.

    2018-03-01

    Molecular cloning of five oxidosqualene cyclases (OSC) genes from Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa had previously been cloned, characterized, and encoded mono and -multi triterpene synthases. The present study analyzed protein modelling of triterpene synthase genes from mangrove using Phyre2 and Swiss-model. The diversity was noted within protein modelling of triterpene synthases using Phyre2 from sequence identity (38-43%) and residue (696-703). RsM2 was distinguishable from others for template structure; it used lanosterol synthase as a template (PDB ID: w6j.1.A). By contrast, other genes used human lanosterol synthase (1w6k.1.A). The predicted bind sites were correlated with the product of triterpene synthase, the product of BgbAS was β-amyrin, while RsM1 contained a significant amount of β-amyrin. Similarly BgLUS and KcMS, both main products was lupeol, on the other hand, RsM2 with the outcome of taraxerol. Homology modelling revealed that 696 residues of BgbAS, BgLUS, RsM1, and RsM2 (91-92% of the amino acid sequence) had been modelled with 100% confidence by the single highest scoring template using Phyre2. This coverage was higher than Swiss-model (85-90%). The present study suggested that molecular cloning of triterpene genes provides useful tools for studying the protein modelling related regulation of isoprenoids biosynthesis in mangrove forests.

  14. ATP Synthase β-Chain Overexpression in SR-BI Knockout Mice Increases HDL Uptake and Reduces Plasma HDL Level

    Directory of Open Access Journals (Sweden)

    Kexiu Song

    2014-01-01

    Full Text Available HDL cholesterol is known to be inversely correlated with cardiovascular disease due to its diverse antiatherogenic functions. SR-BI mediates the selective uptake of HDL-C. SR-BI knockout diminishes but does not completely block the transport of HDL; other receptors may be involved. Ectopic ATP synthase β-chain in hepatocytes has been previously characterized as an apoA-I receptor, triggering HDL internalization. This study was undertaken to identify the overexpression of ectopic ATP synthase β-chain on DIL-HDL uptake in primary hepatocytes in vitro and on plasma HDL levels in SR-BI knockout mice. Human ATP synthase β-chain cDNA was delivered to the mouse liver by adenovirus and GFP adenovirus as control. The adenovirus-mediated overexpression of β-chain was identified at both mRNA and protein levels on mice liver and validated by its increasing of DiL-HDL uptake in primary hepatocytes. In response to hepatic overexpression of β-chain, plasma HDL-C levels and cholesterol were reduced in SR-BI knockout mice, compared with the control. The present data suggest that ATP synthase β-chain can serve as the endocytic receptor of HDL, and its overexpression can reduce plasma HDL-C.

  15. A maize spermine synthase 1 PEST sequence fused to the GUS reporter protein facilitates proteolytic degradation.

    Science.gov (United States)

    Maruri-López, Israel; Rodríguez-Kessler, Margarita; Rodríguez-Hernández, Aída Araceli; Becerra-Flora, Alicia; Olivares-Grajales, Juan Elías; Jiménez-Bremont, Juan Francisco

    2014-05-01

    Polyamines are low molecular weight aliphatic compounds involved in various biochemical, cellular and physiological processes in all organisms. In plants, genes involved in polyamine biosynthesis and catabolism are regulated at transcriptional, translational, and posttranslational level. In this research, we focused on the characterization of a PEST sequence (rich in proline, glutamic acid, serine, and threonine) of the maize spermine synthase 1 (ZmSPMS1). To this aim, 123 bp encoding 40 amino acids of the C-terminal region of the ZmSPMS1 enzyme containing the PEST sequence were fused to the GUS reporter gene. This fusion was evaluated in Arabidopsis thaliana transgenic lines and onion monolayers transient expression system. The ZmSPMS1 PEST sequence leads to specific degradation of the GUS reporter protein. It is suggested that the 26S proteasome may be involved in GUS::PEST fusion degradation in both onion and Arabidopsis. The PEST sequences appear to be present in plant spermine synthases, mainly in monocots. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  16. Citrate synthase proteins in extremophilic organisms: Studies within a structure-based model

    International Nuclear Information System (INIS)

    Różycki, Bartosz; Cieplak, Marek

    2014-01-01

    We study four citrate synthase homodimeric proteins within a structure-based coarse-grained model. Two of these proteins come from thermophilic bacteria, one from a cryophilic bacterium and one from a mesophilic organism; three are in the closed and two in the open conformations. Even though the proteins belong to the same fold, the model distinguishes the properties of these proteins in a way which is consistent with experiments. For instance, the thermophilic proteins are more stable thermodynamically than their mesophilic and cryophilic homologues, which we observe both in the magnitude of thermal fluctuations near the native state and in the kinetics of thermal unfolding. The level of stability correlates with the average coordination number for amino acid contacts and with the degree of structural compactness. The pattern of positional fluctuations along the sequence in the closed conformation is different than in the open conformation, including within the active site. The modes of correlated and anticorrelated movements of pairs of amino acids forming the active site are very different in the open and closed conformations. Taken together, our results show that the precise location of amino acid contacts in the native structure appears to be a critical element in explaining the similarities and differences in the thermodynamic properties, local flexibility, and collective motions of the different forms of the enzyme

  17. Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

    DEFF Research Database (Denmark)

    Højlund, Kurt; Wrzesinski, Krzysztof; Larsen, Peter Mose

    2003-01-01

    quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting...... synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins...

  18. Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

    Science.gov (United States)

    Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Eichler, Duane C

    2012-07-27

    Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

  19. Proteomic profiling of cellulase-aid-extracted membrane proteins for functional identification of cellulose synthase complexes and their potential associated- components in cotton fibers.

    Science.gov (United States)

    Li, Ao; Wang, Ruyi; Li, Xianliang; Liu, Mingyong; Fan, Jian; Guo, Kai; Luo, Bing; Chen, Tingting; Feng, Shengqiu; Wang, Yanting; Wang, Bingrui; Peng, Liangcai; Xia, Tao

    2016-05-19

    Cotton fibers are an excellent model for understanding of cellulose biosynthesis in higher plants. In this study, we determined a high cellulose biosynthesis activity in vitro by optimizing biochemical reaction conditions in cotton fibers. By adding a commercial cellulase enzyme into fibers extraction process, we extracted markedly higher levels of GhCESA1 and GhCESA8 proteins and observed an increase in β-1,4-glucan and β-1,3-glucan products in vitro. LC-MS/MS analysis of anti-GhCESA8-immunoprecipitated proteins showed that 19 proteins could be found in three independent experiments including four CESAs (GhCESA1,2,7,8), five well-known non-CESA proteins, one callose synthase (CALS) and nine novel proteins. Notably, upon the cellulase treatment, four CESAs, one CALS and four novel proteins were measured at relatively higher levels by calculating total peptide counts and distinct peptide numbers, indicating that the cellulase-aid-extracted proteins most likely contribute to the increase in β-glucan products in vitro. These results suggest that the cellulase treatment may aid to release active cellulose synthases complexes from growing glucan chains and make them more amenable to extraction. To our knowledge, it is the first time report about the functional identification of the potential proteins that were associated with plant cellulose and callose synthases complexes by using the cellulase-aided protein extraction.

  20. Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes.

    Science.gov (United States)

    González-Thuillier, Irene; Venegas-Calerón, Mónica; Sánchez, Rosario; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

    2016-02-01

    Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two β-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.

  1. Investigation on oxidative stress of nitric oxide synthase interacting protein from Clonorchis sinensis.

    Science.gov (United States)

    Bian, Meng; Xu, Qingxia; Xu, Yanquan; Li, Shan; Wang, Xiaoyun; Sheng, Jiahe; Wu, Zhongdao; Huang, Yan; Yu, Xinbing

    2016-01-01

    Numerous evidences indicate that excretory-secretory products (ESPs) from liver flukes trigger the generation of free radicals that are associated with the initial pathophysiological responses in host cells. In this study, we first constructed a Clonorchis sinensis (C. sinensis, Cs)-infected BALB/c mouse model and examined relative results respectively at 3, 5, 7, and 9 weeks postinfection (p.i.). Quantitative reverse transcription (RT)-PCR indicated that the transcriptional level of both endothelial nitric oxide synthase (eNOS) and superoxide dismutase (SOD) gradually decreased with lastingness of infection, while the transcriptional level of inducible NOS (iNOS) significantly increased. The level of malondialdehyde (MDA) in sera of infected mouse significantly increased versus the healthy control group. These results showed that the liver of C. sinensis-infected mouse was in a state with elevated levels of oxidation stress. Previously, C. sinensis NOS interacting protein coding gene (named CsNOSIP) has been isolated and recombinant CsNOSIP (rCsNOSIP) has been expressed in Escherichia coli, which has been confirmed to be a component present in CsESPs and confirmed to play important roles in immune regulation of the host. In the present paper, we investigated the effects of rCsNOSIP on the lipopolysaccharide (LPS)-induced activated RAW264.7, a murine macrophage cell line. We found that endotoxin-free rCsNOSIP significantly promoted the levels of nitric oxide (NO) and reactive oxygen species (ROS) after pretreated with rCsNOSIP, while the level of SOD decreased. Furthermore, rCsNOSIP could also increase the level of lipid peroxidation MDA. Taken together, these results suggested that CsNOSIP was a key molecule which was involved in the production of nitric oxide (NO) and its reactive intermediates, and played an important role in oxidative stress during C. sinensis infection.

  2. The Dictyostelium discoideum cellulose synthase: Structure/function analysis and identification of interacting proteins

    Energy Technology Data Exchange (ETDEWEB)

    Richard L. Blanton

    2004-02-19

    OAK-B135 The major accomplishments of this project were: (1) the initial characterization of dcsA, the gene for the putative catalytic subunit of cellulose synthase in the cellular slime mold Dictyostelium discoideum; (2) the detection of a developmentally regulated event (unidentified, but perhaps a protein modification or association with a protein partner) that is required for cellulose synthase activity (i.e., the dcsA product is necessary, but not sufficient for cellulose synthesis); (3) the continued exploration of the developmental context of cellulose synthesis and DcsA; (4) the isolation of a GFP-DcsA-expressing strain (work in progress); and (5) the identification of Dictyostelium homologues for plant genes whose products play roles in cellulose biosynthesis. Although our progress was slow and many of our results negative, we did develop a number of promising avenues of investigation that can serve as the foundation for future projects.

  3. Uridine 5'-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia

    Science.gov (United States)

    Santoso; Thornburg

    1998-02-01

    To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5'-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells.

  4. Uridine 5′-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia1

    Science.gov (United States)

    Santoso, Djoko; Thornburg, Robert

    1998-01-01

    To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5′-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells. PMID:9490773

  5. Wounding stimulates ALLENE OXIDE SYNTHASE gene and increases the level of jasmonic acid in Ipomoea nil cotyledons

    Directory of Open Access Journals (Sweden)

    Emilia Wilmowicz

    2016-03-01

    Full Text Available Allene oxide synthase (AOS encodes the first enzyme in the lipoxygenase pathway, which is responsible for jasmonic acid (JA formation. In this study we report the molecular cloning and characterization of InAOS from Ipomoea nil. The full-length gene is composed of 1662 bp and encodes for 519 amino acids. The predicted InAOS contains PLN02648 motif, which is evolutionarily conserved and characteristic for functional enzymatic proteins. We have shown that wounding led to a strong stimulation of the examined gene activity in cotyledons and an increase in JA level, which suggest that this compound may be a modulator of stress responses in I. nil.

  6. Allene oxide synthase, allene oxide cyclase and jasmonic acid levels in Lotus japonicus nodules.

    Directory of Open Access Journals (Sweden)

    Anna Zdyb

    Full Text Available Jasmonic acid (JA, its derivatives and its precursor cis-12-oxo phytodienoic acid (OPDA form a group of phytohormones, the jasmonates, representing signal molecules involved in plant stress responses, in the defense against pathogens as well as in development. Elevated levels of JA have been shown to play a role in arbuscular mycorrhiza and in the induction of nitrogen-fixing root nodules. In this study, the gene families of two committed enzymes of the JA biosynthetic pathway, allene oxide synthase (AOS and allene oxide cyclase (AOC, were characterized in the determinate nodule-forming model legume Lotus japonicus JA levels were to be analysed in the course of nodulation. Since in all L. japonicus organs examined, JA levels increased upon mechanical disturbance and wounding, an aeroponic culture system was established to allow for a quick harvest, followed by the analysis of JA levels in whole root and shoot systems. Nodulated plants were compared with non-nodulated plants grown on nitrate or ammonium as N source, respectively, over a five week-period. JA levels turned out to be more or less stable independently of the growth conditions. However, L. japonicus nodules formed on aeroponically grown plants often showed patches of cells with reduced bacteroid density, presumably a stress symptom. Immunolocalization using a heterologous antibody showed that the vascular systems of these nodules also seemed to contain less AOC protein than those of nodules of plants grown in perlite/vermiculite. Hence, aeroponically grown L. japonicus plants are likely to be habituated to stress which could have affected JA levels.

  7. Acetylcholine receptor (AChR) clustering is regulated both by glycogen synthase kinase 3β (GSK3β)-dependent phosphorylation and the level of CLIP-associated protein 2 (CLASP2) mediating the capture of microtubule plus-ends.

    Science.gov (United States)

    Basu, Sreya; Sladecek, Stefan; Pemble, Hayley; Wittmann, Torsten; Slotman, Johan A; van Cappellen, Wiggert; Brenner, Hans-Rudolf; Galjart, Niels

    2014-10-31

    The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2-9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2-8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. A cellulose synthase-like protein is required for osmotic stress tolerance in Arabidopsis

    KAUST Repository

    Zhu, Jianhua

    2010-04-16

    Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root-bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6-1, which defines a locus essential for osmotic stress tolerance. sos6-1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase-like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6-1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress. © 2010 Blackwell Publishing Ltd.

  9. Strategies in megasynthase engineering – fatty acid synthases (FAS as model proteins

    Directory of Open Access Journals (Sweden)

    Manuel Fischer

    2017-06-01

    Full Text Available Megasynthases are large multienzyme proteins that produce a plethora of important natural compounds by catalyzing the successive condensation and modification of precursor units. Within the class of megasynthases, polyketide synthases (PKS are responsible for the production of a large spectrum of bioactive polyketides (PK, which have frequently found their way into therapeutic applications. Rational engineering approaches have been performed during the last 25 years that seek to employ the “assembly-line synthetic concept” of megasynthases in order to deliver new bioactive compounds. Here, we highlight PKS engineering strategies in the light of the newly emerging structural information on megasynthases, and argue that fatty acid synthases (FAS are and will be valuable objects for further developing this field.

  10. Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

    Directory of Open Access Journals (Sweden)

    Helena H. Chowdhury

    2014-10-01

    Full Text Available Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

  11. A recruiting protein of geranylgeranyl diphosphate synthase controls metabolic flux toward chlorophyll biosynthesis in rice.

    Science.gov (United States)

    Zhou, Fei; Wang, Cheng-Yuan; Gutensohn, Michael; Jiang, Ling; Zhang, Peng; Zhang, Dabing; Dudareva, Natalia; Lu, Shan

    2017-06-27

    In plants, geranylgeranyl diphosphate (GGPP) is produced by plastidic GGPP synthase (GGPPS) and serves as a precursor for vital metabolic branches, including chlorophyll, carotenoid, and gibberellin biosynthesis. However, molecular mechanisms regulating GGPP allocation among these biosynthetic pathways localized in the same subcellular compartment are largely unknown. We found that rice contains only one functionally active GGPPS, OsGGPPS1, in chloroplasts. A functionally active homodimeric enzyme composed of two OsGGPPS1 subunits is located in the stroma. In thylakoid membranes, however, the GGPPS activity resides in a heterodimeric enzyme composed of one OsGGPPS1 subunit and GGPPS recruiting protein (OsGRP). OsGRP is structurally most similar to members of the geranyl diphosphate synthase small subunit type II subfamily. In contrast to members of this subfamily, OsGRP enhances OsGGPPS1 catalytic efficiency and specificity of GGPP production on interaction with OsGGPPS1. Structural biology and protein interaction analyses demonstrate that affinity between OsGRP and OsGGPPS1 is stronger than between two OsGGPPS1 molecules in homodimers. OsGRP determines OsGGPPS1 suborganellar localization and directs it to a large protein complex in thylakoid membranes, consisting of geranylgeranyl reductase (OsGGR), light-harvesting-like protein 3 (OsLIL3), protochlorophyllide oxidoreductase (OsPORB), and chlorophyll synthase (OsCHLG). Taken together, genetic and biochemical analyses suggest OsGRP functions in recruiting OsGGPPS1 from the stroma toward thylakoid membranes, thus providing a mechanism to control GGPP flux toward chlorophyll biosynthesis.

  12. The role of oligomerization and cooperative regulation in protein function: the case of tryptophan synthase.

    Directory of Open Access Journals (Sweden)

    M Qaiser Fatmi

    Full Text Available The oligomerization/co-localization of protein complexes and their cooperative regulation in protein function is a key feature in many biological systems. The synergistic regulation in different subunits often enhances the functional properties of the multi-enzyme complex. The present study used molecular dynamics and Brownian dynamics simulations to study the effects of allostery, oligomerization and intermediate channeling on enhancing the protein function of tryptophan synthase (TRPS. TRPS uses a set of α/β-dimeric units to catalyze the last two steps of L-tryptophan biosynthesis, and the rate is remarkably slower in the isolated monomers. Our work shows that without their binding partner, the isolated monomers are stable and more rigid. The substrates can form fairly stable interactions with the protein in both forms when the protein reaches the final ligand-bound conformations. Our simulations also revealed that the α/β-dimeric unit stabilizes the substrate-protein conformation in the ligand binding process, which lowers the conformation transition barrier and helps the protein conformations shift from an open/inactive form to a closed/active form. Brownian dynamics simulations with a coarse-grained model illustrate how protein conformations affect substrate channeling. The results highlight the complex roles of protein oligomerization and the fine balance between rigidity and dynamics in protein function.

  13. Novel protein–protein interaction between spermidine synthase and S-adenosylmethionine decarboxylase from Leishmania donovani

    Energy Technology Data Exchange (ETDEWEB)

    Mishra, Arjun K.; Agnihotri, Pragati; Srivastava, Vijay Kumar; Pratap, J. Venkatesh, E-mail: jvpratap@cdri.res.in

    2015-01-09

    Highlights: • L. donovani spermidine synthase and S-adenosylmethionine decarboxylase have been cloned and purified. • S-adenosylmethionine decarboxylase has autocatalytic property. • GST pull down assay shows the two proteins to form a metabolon. • Isothermal titration calorimetry shows that binding was exothermic having K{sub d} value of 0.4 μM. • Interaction confirmed by fluorescence spectroscopy and size exclusion chromatography. - Abstract: Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein–protein interaction between SpdSyn and AdoMetDc. The protein–protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes.

  14. Constitutive expression of feedback-insensitive cystathionine γ-synthase increases methionine levels in soybean leaves and seeds

    Institute of Scientific and Technical Information of China (English)

    YU Yang; HOU Wen-sheng; YaeI Hacham; SUN Shi; WU Cun-xiang; Ifat Matityahu; SONG Shikui; RacheI Amir; HAN Tian-fu

    2018-01-01

    Soybean (Glycine max (L.) Merr.) is a major crop that provides plant-origin protein and oil for humans and livestock. Although the soybean vegetative tissues and seeds provide a major source of high-quality protein, they suffer from low concentration of an essential sulfur-containing amino acid, methionine, which significantly limits their nutritional quality. The level of methionine is mainly controlled by the first unique enzyme of methionine synthesis, cystathione γ-synthase (CGS). Aiming to elevate methionine level in vegetative tissues and seeds, we constitutively over-expressed a feedback-insensitive Arabidopsis CGS (AtD-CGS) in soybean cultivars, Zigongdongdou (ZD) and Jilinxiaoli 1 (JX). The levels of soluble methionine increased remarkably in leaves of transgenic soybeans compared to wild-type plants (6.6- and 7.3-fold in two transgenic ZD lines, and 3.7-fold in one transgenic JX line). Furthermore, the total methionine contents were significantly increased in seeds of the transgenic ZD lines (1.5- to 4.8-fold increase) and the transgenic JX lines (1.3- to 2.3-fold increase) than in the wild type. The protein contents of the transgenic soybean seeds were significantly elevated compared to the wild type, suggesting that the scarcity of methionine in soybeans may limit protein accumulation in soybean seeds. The increased protein content did not alter the profile of major storage proteins in the seeds. Generally, this study provides a promising strategy to increase the levels of methionine and protein in soybean through the breeding programs.

  15. Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yechun; Yi, Hankuil; Wang, Melissa; Yu, Oliver; Jez, Joseph M. (WU); (Danforth)

    2012-10-24

    To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the first molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 {angstrom} of each other provides the basis for enhanced in vivo synthesis of resveratrol.

  16. [PREPARATION OF HUMAN TISSUE PROTEIN EXTRACTS ENRICHED WITH THE SPHINGOMYELIN SYNTHASE 1].

    Science.gov (United States)

    Sudarkina, O Yu; Dergunova, L V

    2015-01-01

    Sphingomyelin synthase 1 (SMS 1) catalyzes sphingomyelin biosynthesis in eukaryotic cells. We previously studied the structure of the human SGMS1 gene, which encodes the enzyme and its numerous transcripts. The tissue-specific expression of the transcripts was also described. Analysis of the SMS1 protein expression in human tissues using immunoblotting of tissue extracts prepared in the RIPA (Radio Immuno-Precipitation Assay) buffer revealed a weak signal in renal cortex, testis, lung, and no signal in placenta and lymphatic node. In this work, a new method of preparation of the tissue protein extracts enriched with SMS1 was suggested. The method based on the consecutive extraction with a buffer containing 0.05 and 1 mg/ml of the Quillaja saponaria saponin allowed SMS1 to be detected in all tissues tested. The SMS1 content in the saponin extract of kidney cortex is about 12-fold higher compared to the RIPA extraction procedure.

  17. Role of heat shock protein 90 and endothelial nitric oxide synthase during early anesthetic and ischemic preconditioning.

    Science.gov (United States)

    Amour, Julien; Brzezinska, Anna K; Weihrauch, Dorothee; Billstrom, Amie R; Zielonka, Jacek; Krolikowski, John G; Bienengraeber, Martin W; Warltier, David C; Pratt, Philip F; Kersten, Judy R

    2009-02-01

    Nitric oxide is known to be essential for early anesthetic preconditioning (APC) and ischemic preconditioning (IPC) of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, the authors tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC. Myocardial infarct size was measured in rabbits after coronary occlusion and reperfusion in the absence or presence of preconditioning within 30 min of isoflurane (APC) or 5 min of coronary artery occlusion (IPC), and with or without pretreatment with geldanamycin or radicicol, two chemically distinct Hsp90 inhibitors, or N-nitro-L-arginine methyl ester, a nonspecific nitric oxide synthase NOS inhibitor. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells or mouse cardiomyocytes, in the absence or presence of Hsp90 inhibitors or N-nitro-L-arginine methyl ester. Interactions between Hsp90 and eNOS, and eNOS activation, were assessed with immunoprecipitation, immunoblotting, and confocal microscopy. APC and IPC decreased infarct size (by 50% and 59%, respectively), and this action was abolished by Hsp90 inhibitors. N-nitro-L-arginine methyl ester blocked APC but not IPC. Isoflurane increased nitric oxide production in human coronary artery endothelial cells concomitantly with an increase in Hsp90-eNOS interaction (immunoprecipitation, immunoblotting, and immunohistochemistry). Pretreatment with Hsp90 inhibitors abolished isoflurane-dependent nitric oxide production and decreased Hsp90-eNOS interactions. Isoflurane did not increase nitric oxide production in mouse cardiomyocytes, and eNOS was below the level of detection. The results indicate that Hsp90 plays a critical role in mediating APC and IPC through protein-protein interactions, and suggest that endothelial cells are important contributors to nitric oxide-mediated signaling during APC.

  18. Molecular cloning and expression levels of the monoterpene synthase gene (ZMM1 in Cassumunar ginger (Zingiber montanum (Koenig Link ex Dietr.

    Directory of Open Access Journals (Sweden)

    Bua-In Saowaluck

    2014-01-01

    Full Text Available Cassumunar ginger (Zingiber montanum (Koenig Link ex Dietr. is a native Thai herb with a high content and large variety of terpenoids in its essential oil. Improving the essential oil content and quality of cassumunar ginger is difficult for a breeder due to its clonally propagated nature. In this research, we describe the isolation and expression level of the monoterpene synthase gene that controls the key step of essential oil synthesis in this plant and evaluate the mechanical wounding that may influence the transcription level of the monoterpene synthase gene. To isolate the gene, the selected clones from DNA derived from young leaves were sequenced and analyzed and the monoterpene synthase gene from cassumunar ginger (ZMM1 was identified. The ZMM1 CDS containing 1 773 bp (KF500399 is predicted to encode a protein of 590 amino acids. The deduced amino acid sequence is 40-74% identical with known sequences of other angiosperm monoterpene synthases belonging to the isoprenoid biosynthesis C1 superfamily. A transcript of ZMM1 was detected almost exclusively in the leaves and was related to leaf wounding. The results of this research offer insight into the control of monoterpene synthesis in this plant. This finding can be applied to breeding programs or crop management of cassumunar ginger for better yield and quality of essential oil.

  19. Identification, immunolocalization, and immunological characterization of nitric oxide synthase-interacting protein from Clonorchis sinensis.

    Science.gov (United States)

    Bian, Meng; Li, Shan; Wang, Xiaoyun; Xu, Yanquan; Chen, Wenjun; Zhou, Chenhui; Chen, Xueqing; He, Lei; Xu, Jin; Liang, Chi; Wu, Zhongdao; Huang, Yan; Li, Xuerong; Yu, Xinbing

    2014-05-01

    Recently, accumulating evidences indicate that nitric oxide (NO) is a potent mediator with diverse roles in regulating cellular functions, signaling pathways, and variety of pathological processes. In the present study, using data from the published genomic for Clonorchis sinensis (C. sinensis), we investigated a gene encoding nitric oxide synthase-interacting protein (NOSIP) of C. sinensis. Recombinant CsNOSIP (rCsNOSIP) was expressed and purified from Escherichia coli BL21. The open reading frame of CsNOSIP comprises 867 bp which encodes 289 amino acids and shares 72.9, 45.2, 47, 46.4, and 45.8% identity with NOSIP from Schistosoma mansoni, Xenopus laevis, Rattus norvegicus, Mus musculus, and Homo sapiens, respectively. Bioinformatics analysis suggested that the full-length sequence contains an eNOS-interacting domain and numerous B-cell epitopes. Quantitative RT-PCR indicated that CsNOSIP differentially transcribed throughout the adult worms, metacercariae, and egg stages of C. sinensis, and were highly expressed in the adult worms. Moreover, western blot analysis showed that the rCsNOSIP could be detected by the serum from BALB/c mice infected with C. sinensis and the serum from BALB/c mice immunized with excretory/secretory products (ESPs). Furthermore, immunolocalization assay showed that CsNOSIP was specifically localized in the intestine, vitellarium, and eggs of adult worm. Both immunoblot and immunolocalization results demonstrated that CsNOSIP was one component of ESPs of C. sinensis, which could be supported by SignalP analysis. Moreover, analysis of the antibody subclass and cytokine profile demonstrated that subcutaneously immunized BALB/c mice with rCsNOSIP could significantly enhance serum IgG1 level and up-regulate expression of IL-4 and IL-6 in the splenocytes. Our results suggested that CsNOSIP was an important antigen exposed to host immune system and probably involved in immune regulation of host by inducing Th2-polarized immune response.

  20. C75, a fatty acid synthase inhibitor, modulates AMP-activated protein kinase to alter neuronal energy metabolism.

    Science.gov (United States)

    Landree, Leslie E; Hanlon, Andrea L; Strong, David W; Rumbaugh, Gavin; Miller, Ian M; Thupari, Jagan N; Connolly, Erin C; Huganir, Richard L; Richardson, Christine; Witters, Lee A; Kuhajda, Francis P; Ronnett, Gabriele V

    2004-01-30

    C75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior.

  1. Geranylgeranyl diphosphate synthase in fission yeast is a heteromer of farnesyl diphosphate synthase (FPS), Fps1, and an FPS-like protein, Spo9, essential for sporulation.

    Science.gov (United States)

    Ye, Yanfang; Fujii, Makoto; Hirata, Aiko; Kawamukai, Makoto; Shimoda, Chikashi; Nakamura, Taro

    2007-09-01

    Both farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) are key enzymes in the synthesis of various isoprenoid-containing compounds and proteins. Here, we describe two novel Schizosaccharomyces pombe genes, fps1(+) and spo9(+), whose products are similar to FPS in primary structure, but whose functions differ from one another. Fps1 is essential for vegetative growth, whereas, a spo9 null mutant exhibits temperature-sensitive growth. Expression of fps1(+), but not spo9(+), suppresses the lethality of a Saccharomyces cerevisiae FPS-deficient mutant and also restores ubiquinone synthesis in an Escherichia coli ispA mutant, which lacks FPS activity, indicating that S. pombe Fps1 in fact functions as an FPS. In contrast to a typical FPS gene, no apparent GGPS homologues have been found in the S. pombe genome. Interestingly, although neither fps1(+) nor spo9(+) expression alone in E. coli confers clear GGPS activity, coexpression of both genes induces such activity. Moreover, the GGPS activity is significantly reduced in the spo9 mutant. In addition, the spo9 mutation perturbs the membrane association of a geranylgeranylated protein, but not that of a farnesylated protein. Yeast two-hybrid and coimmunoprecipitation analyses indicate that Fps1 and Spo9 physically interact. Thus, neither Fps1 nor Spo9 alone functions as a GGPS, but the two proteins together form a complex with GGPS activity. Because spo9 was originally identified as a sporulation-deficient mutant, we show here that expansion of the forespore membrane is severely inhibited in spo9Delta cells. Electron microscopy revealed significant accumulation membrane vesicles in spo9Delta cells. We suggest that lack of GGPS activity in a spo9 mutant results in impaired protein prenylation in certain proteins responsible for secretory function, thereby inhibiting forespore membrane formation.

  2. Glycogen synthase kinase 3β promotes liver innate immune activation by restraining AMP-activated protein kinase activation.

    Science.gov (United States)

    Zhou, Haoming; Wang, Han; Ni, Ming; Yue, Shi; Xia, Yongxiang; Busuttil, Ronald W; Kupiec-Weglinski, Jerzy W; Lu, Ling; Wang, Xuehao; Zhai, Yuan

    2018-02-13

    Glycogen synthase kinase 3β (Gsk3β [Gsk3b]) is a ubiquitously expressed kinase with distinctive functions in different types of cells. Although its roles in regulating innate immune activation and ischaemia and reperfusion injuries (IRIs) have been well documented, the underlying mechanisms remain ambiguous, in part because of the lack of cell-specific tools in vivo. We created a myeloid-specific Gsk3b knockout (KO) strain to study the function of Gsk3β in macrophages in a murine liver partial warm ischaemia model. Compared with controls, myeloid Gsk3b KO mice were protected from IRI, with diminished proinflammatory but enhanced anti-inflammatory immune responses in livers. In bone marrow-derived macrophages, Gsk3β deficiency resulted in an early reduction of Tnf gene transcription but sustained increase of Il10 gene transcription on Toll-like receptor 4 stimulation in vitro. These effects were associated with enhanced AMP-activated protein kinase (AMPK) activation, which led to an accelerated and higher level of induction of the novel innate immune negative regulator small heterodimer partner (SHP [Nr0b2]). The regulatory function of Gsk3β on AMPK activation and SHP induction was confirmed in wild-type bone marrow-derived macrophages with a Gsk3 inhibitor. Furthermore, we found that this immune regulatory mechanism was independent of Gsk3β Ser9 phosphorylation and the phosphoinositide 3-kinase-Akt signalling pathway. In vivo, myeloid Gsk3β deficiency facilitated SHP upregulation by ischaemia-reperfusion in liver macrophages. Treatment of Gsk3b KO mice with either AMPK inhibitor or SHP small interfering RNA before the onset of liver ischaemia restored liver proinflammatory immune activation and IRI in these otherwise protected hosts. Additionally, pharmacological activation of AMPK protected wild-type mice from liver IRI, with reduced proinflammatory immune activation. Inhibition of the AMPK-SHP pathway by liver ischaemia was demonstrated in tumour resection

  3. Arabidopsis OR proteins are the major post-transcriptional regulators of phytoene synthase in mediating carotenoid biosynthesis

    Science.gov (United States)

    Carotenoids are indispensable natural pigments to plants and humans. Phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthetic pathway, and ORANGE (OR), a regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis, represent two key proteins that control caro...

  4. Thymidylate Synthase, Thymidine Phosphorylase and Orotate Phosphoribosyl Transferase Levels as Predictive Factors of Chemotherapy in Oral Squamous Cell Carcinoma

    International Nuclear Information System (INIS)

    Ogiuchi, Yosuke; Maruoka, Yasubumi; Ando, Tomohiro; Kobayashi, Makio; Ogiuchi, Hideki

    2008-01-01

    We conducted a clinicopathologic study on protein and mRNA levels of thymidylate synthase (TS), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT) using biopsy tissue specimens before treatment. The mRNA levels have been measured in tumor cells microdissected from paraffin-embedded specimens (Danenberg Tumor Profile method: DTP method). We studied the mRNA and protein expression as effect predictive factors in chemotherapy. The subjects consisted of 20 cases of untreated oral squamous cell carcinoma who had undergone chemotherapy with TS-1 (16 males and 4 females, tongue in 8 cases, upper gingiva in 3 cases, lower gingiva in 3 cases, buccal mucosa in 5 cases and floor of the mouth in 1 case). TS gene expressions of the responders were lower than those for the nonresponders. Furthermore, regarding males who were less than 70 years of age, stage I and II, well differentiated type and tongue, TS mRNA expression of the responders were lower than that for the nonresponders. The mRNA expression of OPRT for the male responders was lower than that for the nonresponders. No remarkable difference was observed by immunohistochemistry. In this study, the measurement of the TS levels using the DTP method may potentially act as a predictive factor of antitumor effectiveness

  5. Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Halavaty, Andrei S. [Center for Structural Genomics of Infectious Diseases, (United States); Northwestern University, Chicago, IL 60611 (United States); Kim, Youngchang [Center for Structural Genomics of Infectious Diseases, (United States); Argonne National Laboratory, Argonne, IL 60439 (United States); University of Chicago, Chicago, IL 60637 (United States); Minasov, George; Shuvalova, Ludmilla; Dubrovska, Ievgeniia; Winsor, James [Center for Structural Genomics of Infectious Diseases, (United States); Northwestern University, Chicago, IL 60611 (United States); Zhou, Min [Center for Structural Genomics of Infectious Diseases, (United States); Argonne National Laboratory, Argonne, IL 60439 (United States); University of Chicago, Chicago, IL 60637 (United States); Onopriyenko, Olena; Skarina, Tatiana [Center for Structural Genomics of Infectious Diseases, (United States); University of Toronto, Toronto, Ontario M5G 1L6 (Canada); Papazisi, Leka; Kwon, Keehwan; Peterson, Scott N. [Center for Structural Genomics of Infectious Diseases, (United States); J. Craig Venter Institute, Rockville, MD 20850 (United States); Joachimiak, Andrzej [Center for Structural Genomics of Infectious Diseases, (United States); Argonne National Laboratory, Argonne, IL 60439 (United States); University of Chicago, Chicago, IL 60637 (United States); Savchenko, Alexei [Center for Structural Genomics of Infectious Diseases, (United States); University of Toronto, Toronto, Ontario M5G 1L6 (Canada); Anderson, Wayne F., E-mail: wf-anderson@northwestern.edu [Center for Structural Genomics of Infectious Diseases, (United States); Northwestern University, Chicago, IL 60611 (United States)

    2012-10-01

    The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpS{sub SA}), Vibrio cholerae (AcpS{sub VC}) and Bacillus anthracis (AcpS{sub BA}) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpS{sub BA} is emphasized because of the two 3′, 5′-adenosine diphosphate (3′, 5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′, 5′-ADP is bound as the 3′, 5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′, 5′-ADP–AcpS binary complexes. The position of the second 3′, 5′-ADP has never been described before. It is in close proximity to the first 3′, 5′-ADP and the ACP-binding site. The coordination of two ADPs in AcpS{sub BA} may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.

  6. Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria

    International Nuclear Information System (INIS)

    Halavaty, Andrei S.; Kim, Youngchang; Minasov, George; Shuvalova, Ludmilla; Dubrovska, Ievgeniia; Winsor, James; Zhou, Min; Onopriyenko, Olena; Skarina, Tatiana; Papazisi, Leka; Kwon, Keehwan; Peterson, Scott N.; Joachimiak, Andrzej; Savchenko, Alexei; Anderson, Wayne F.

    2012-01-01

    The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpS SA ), Vibrio cholerae (AcpS VC ) and Bacillus anthracis (AcpS BA ) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpS BA is emphasized because of the two 3′, 5′-adenosine diphosphate (3′, 5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′, 5′-ADP is bound as the 3′, 5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′, 5′-ADP–AcpS binary complexes. The position of the second 3′, 5′-ADP has never been described before. It is in close proximity to the first 3′, 5′-ADP and the ACP-binding site. The coordination of two ADPs in AcpS BA may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP

  7. A phycocyanin·phellandrene synthase fusion enhances recombinant protein expression and β-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria).

    Science.gov (United States)

    Formighieri, Cinzia; Melis, Anastasios

    2015-11-01

    Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of Synechocystis and heterologous expression of the β-phellandrene synthase (PHLS) gene alone is necessary and sufficient to confer to Synechocystis the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow Kcat (low Vmax) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-phellandrene product generation. Different PHLS fusion constructs were generated with the Synechocystis endogenous cpcB sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native cpc operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous cpc promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-phellandrene synthesis, yielding an average of 3.2 mg product g(-1) dry cell weight (dcw) versus the 0.03 mg g(-1)dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-phellandrene hydrocarbons production in these photosynthetic microorganisms. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  8. Effects of starch synthase IIa gene dosage on grain, protein and starch in endosperm of wheat.

    Science.gov (United States)

    Konik-Rose, Christine; Thistleton, Jenny; Chanvrier, Helene; Tan, Ihwa; Halley, Peter; Gidley, Michael; Kosar-Hashemi, Behjat; Wang, Hong; Larroque, Oscar; Ikea, Joseph; McMaugh, Steve; Regina, Ahmed; Rahman, Sadequr; Morell, Matthew; Li, Zhongyi

    2007-11-01

    Starch synthases (SS) are responsible for elongating the alpha-1,4 glucan chains of starch. A doubled haploid population was generated by crossing a line of wheat, which lacks functional ssIIa genes on each genome (abd), and an Australian wheat cultivar, Sunco, with wild type ssIIa alleles on each genome (ABD). Evidence has been presented previously indicating that the SGP-1 (starch granule protein-1) proteins present in the starch granule in wheat are products of the ssIIa genes. Analysis of 100 progeny lines demonstrated co-segregation of the ssIIa alleles from the three genomes with the SGP-1 proteins, providing further evidence that the SGP-1 proteins are the products of the ssIIa genes. From the progeny lines, 40 doubled haploid lines representing the eight possible genotypes for SSIIa (ABD, aBD, AbD, ABd, abD, aBd, Abd, abd) were characterized for their grain weight, protein content, total starch content and starch properties. For some properties (chain length distribution, pasting properties, swelling power, and gelatinization properties), a progressive change was observed across the four classes of genotypes (wild type, single nulls, double nulls and triple nulls). However, for other grain properties (seed weight and protein content) and starch properties (total starch content, granule morphology and crystallinity, granule size distribution, amylose content, amylose-lipid dissociation properties), a statistically significant change only occurred for the triple nulls, indicating that all three genes had to be missing or inactive for a change to occur. These results illustrate the importance of SSIIa in controlling grain and starch properties and the importance of amylopectin fine structure in controlling starch granule properties in wheat.

  9. Predicted cycloartenol synthase protein from Kandelia obovata and Rhizophora stylosa using online software of Phyre2 and Swiss-model

    Science.gov (United States)

    Basyuni, M.; Sulistiyono, N.; Wati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.

    2018-03-01

    Cloning of Kandelia obovata KcCAS gene (previously known as Kandelia candel) and Rhizophora stylosa RsCAS have already have been reported and encoded cycloartenol synthases. In this study, the predicted KcCAS and RsCAS protein were analyzed using online software of Phyre2 and Swiss-model. The protein modelling for KcCAS and RsCAS cycloartenol synthases was determined using Pyre2 had similar results with slightly different in sequence identity. By contrast, the Swiss-model for KcCAS slightly had higher sequence identity (47.31%) and Qmean (0.70) compared to RsCAS. No difference of ligands binding site which is considered as modulators for both cycloartenol synthases. The range of predicted protein derived from 91-757 amino acid residues with coverage sequence similarities 0.86, respectively from template model of lanosterol synthase from the human. Homology modelling revealed that 706 residues (93% of the amino acid sequence) had been modelled with 100.0% confidence by the single highest scoring template for both KcCAS and RsCAS using Phyre2. This coverage was more elevated than swiss-model predicted (86%). The present study suggested that both genes are responsible for the genesis of cycloartenol in these mangrove plants.

  10. 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.

  11. Hyperglycemia adversely modulates endothelial nitric oxide synthase during anesthetic preconditioning through tetrahydrobiopterin- and heat shock protein 90-mediated mechanisms.

    Science.gov (United States)

    Amour, Julien; Brzezinska, Anna K; Jager, Zachary; Sullivan, Corbin; Weihrauch, Dorothee; Du, Jianhai; Vladic, Nikolina; Shi, Yang; Warltier, David C; Pratt, Phillip F; Kersten, Judy R

    2010-03-01

    Endothelial nitric oxide synthase activity is regulated by (6R-)5,6,7,8-tetrahydrobiopterin (BH4) and heat shock protein 90. The authors tested the hypothesis that hyperglycemia abolishes anesthetic preconditioning (APC) through BH4- and heat shock protein 90-dependent pathways. Myocardial infarct size was measured in rabbits in the absence or presence of APC (30 min of isoflurane), with or without hyperglycemia, and in the presence or absence of the BH4 precursor sepiapterin. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells cultured in normal (5.5 mm) or high (20 mm) glucose conditions, with or without sepiapterin (10 or 100 microm). APC decreased myocardial infarct size compared with control experiments (26 +/- 6% vs. 46 +/- 3%, respectively; P < 0.05), and this action was blocked by hyperglycemia (43 +/- 4%). Sepiapterin alone had no effect on infarct size (46 +/- 3%) but restored APC during hyperglycemia (21 +/- 3%). The beneficial actions of sepiapterin to restore APC were blocked by the nitric oxide synthase inhibitor N (G)-nitro-L-arginine methyl ester (47 +/- 2%) and the BH4 synthesis inhibitor N-acetylserotonin (46 +/- 3%). Isoflurane increased nitric oxide production to 177 +/- 13% of baseline, and this action was attenuated by high glucose concentrations (125 +/- 6%). Isoflurane increased, whereas high glucose attenuated intracellular BH4/7,8-dihydrobiopterin (BH2) (high performance liquid chromatography), heat shock protein 90-endothelial nitric oxide synthase colocalization (confocal microscopy) and endothelial nitric oxide synthase activation (immunoblotting). Sepiapterin increased BH4/BH2 and dose-dependently restored nitric oxide production during hyperglycemic conditions (149 +/- 12% and 175 +/- 9%; 10 and 100 microm, respectively). The results indicate that tetrahydrobiopterin and heat shock protein 90-regulated endothelial nitric oxide synthase activity play a central

  12. Threonine phosphorylation of rat liver glycogen synthase

    International Nuclear Information System (INIS)

    Arino, J.; Arro, M.; Guinovart, J.J.

    1985-01-01

    32 P-labeled glycogen synthase specifically immunoprecipitated from 32 P-phosphate incubated rat hepatocytes contains, in addition to [ 32 P] phosphoserine, significant levels of [ 32 P] phosphothreonine. When the 32 P-immunoprecipitate was cleaved with CNBr, the [ 32 P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 in vitro. After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32 P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the ''in vivo'' phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase

  13. Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex

    Energy Technology Data Exchange (ETDEWEB)

    Marcella, Aaron M.; Culbertson, Sannie J.; Shogren-Knaak, Michael A.; Barb, Adam W.

    2017-11-01

    The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05 and 4.10 Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a KD = 62 ± 13 nM, followed by the binding of two more equivalents of holo-ACPP with KD = 1.2 ± 0.2 μM. Cooperativity was not observed for apo-ACPP which bound with KD = 2.4 ± 0.1 μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.

  14. Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex.

    Science.gov (United States)

    Marcella, Aaron M; Culbertson, Sannie J; Shogren-Knaak, Michael A; Barb, Adam W

    2017-11-24

    The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05and 4.10Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a K D =62±13nM, followed by the binding of two more equivalents of holo-ACPP with K D =1.2±0.2μM. Cooperativity was not observed for apo-ACPP which bound with K D =2.4±0.1μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. The major antigenic membrane protein of "Candidatus Phytoplasma asteris" selectively interacts with ATP synthase and actin of leafhopper vectors.

    Directory of Open Access Journals (Sweden)

    Luciana Galetto

    Full Text Available Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of "Candidatus Phytoplasma asteris", the chrysanthemum yellows phytoplasmas (CYP strain, and three others as non-vectors. Interactions between a labelled (recombinant CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

  16. Inhibition of neutral sphingomyelinase decreases elevated levels of inducible nitric oxide synthase and apoptotic cell death in ocular hypertensive rats

    Energy Technology Data Exchange (ETDEWEB)

    Aslan, Mutay, E-mail: mutayaslan@akdeniz.edu.tr [Department of Medical Biochemistry, Akdeniz University Faculty of Medicine, Antalya (Turkey); Basaranlar, Goksun [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Unal, Mustafa [Department of Ophthalmology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Ciftcioglu, Akif [Department of Pathology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Derin, Narin [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Mutus, Bulent [Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario (Canada)

    2014-11-01

    Endoplasmic reticulum (ER) stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of neuronal retinal cell death in ocular hypertension. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression, hence this study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on retinal NOS2 levels, ER stress, apoptosis and visual evoked potentials (VEPs) in a rat model of elevated intraocular pressure (EIOP). NOS2 expression and retinal protein nitration were significantly greater in EIOP and significantly decreased with N-SMase inhibition. A significant increase was observed in retinal ER stress markers pPERK, CHOP and GRP78 in EIOP, which were not significantly altered by N-SMase inhibition. Retinal TUNEL staining showed increased apoptosis in all EIOP groups; however N-SMase inhibition significantly decreased the percent of apoptotic cells in EIOP. Caspase-3, -8 and -9 activities were significantly increased in EIOP and returned to baseline levels following N-SMase inhibition. Latencies of all VEP components were significantly prolonged in EIOP and shortened following N-SMase inhibition. Data confirm the role of nitrative injury in EIOP and highlight the protective effect of N-SMase inhibition in EIOP via down-regulation of NOS2 levels and nitrative stress. - Highlights: • Inhibition of N-SMase decreases NOS2 levels in ocular hypertension. • Inhibition of N-SMase decreases protein nitration in ocular hypertension. • Inhibition of N-SMase decreases caspase activation in ocular hypertension. • Inhibition of N-SMase decreases apoptosis in ocular hypertension.

  17. Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

    Directory of Open Access Journals (Sweden)

    Lu Frances Fangjia

    2012-11-01

    Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  18. Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels.

    Science.gov (United States)

    Yang, Ke; Guo, Yang; Stacey, William C; Harwalkar, Jyoti; Fretthold, Jonathan; Hitomi, Masahiro; Stacey, Dennis W

    2006-08-30

    The expression level of cyclin D1 plays a vital role in the control of proliferation. This protein is reported to be degraded following phosphorylation by glycogen synthase kinase 3 (GSK3) on Thr-286. We recently showed that phosphorylation of Thr-286 is responsible for a decline in cyclin D1 levels during S phase, an event required for efficient DNA synthesis. These studies were undertaken to test the possibility that phosphorylation by GSK3 is responsible for the S phase specific decline in cyclin D1 levels, and that this event is regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway which controls GSK3. We found, however, that neither PI3K, AKT, GSK3, nor proliferative signaling activity in general is responsible for the S phase decline in cyclin D1 levels. In fact, the activity of these signaling kinases does not vary through the cell cycle of proliferating cells. Moreover, we found that GSK3 activity has little influence over cyclin D1 expression levels during any cell cycle phase. Inhibition of GSK3 activity by siRNA, LiCl, or other chemical inhibitors failed to influence cyclin D1 phosphorylation on Thr-286, even though LiCl efficiently blocked phosphorylation of beta-catenin, a known substrate of GSK3. Likewise, the expression of a constitutively active GSK3 mutant protein failed to influence cyclin D1 phosphorylation or total protein expression level. Because we were unable to identify any proliferative signaling molecule or pathway which is regulated through the cell cycle, or which is able to influence cyclin D1 levels, we conclude that the suppression of cyclin D1 levels during S phase is regulated by cell cycle position rather than signaling activity. We propose that this mechanism guarantees the decline in cyclin D1 levels during each S phase; and that in so doing it reduces the likelihood that simple over expression of cyclin D1 can lead to uncontrolled cell growth.

  19. Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Peng, Bingyin; Nielsen, Lars K.; Kampranis, Sotirios C

    2018-01-01

    Monoterpene production in Saccharomyces cerevisae requires the introduction of heterologous monoterpene synthases (MTSs). The endogenous farnesyl pyrosphosphate synthase (FPPS; Erg20p) competes with MTSs for the precursor geranyl pyrophosphate (GPP), which limits the production of monoterpenes. ERG......20 is an essential gene that cannot be deleted and transcriptional down-regulation of ERG20 has failed to improve monoterpene production. Here, we investigated an N-degron-dependent protein degradation strategy to down-regulate Erg20p activity. Degron tagging decreased GFP protein half......-life drastically to 1 h (degron K3K15) or 15 min (degrons KN113 and KN119). Degron tagging of ERG20 was therefore paired with a sterol responsive promoter to ensure sufficient metabolic flux to essential downstream sterols despite the severe destabilisation effect of degron tagging. A dual monoterpene...

  20. Glycogen synthase kinase-3 levels and phosphorylation undergo large fluctuations in mouse brain during development

    Science.gov (United States)

    Beurel, Eléonore; Mines, Marjelo A; Song, Ling; Jope, Richard S

    2012-01-01

    Objectives Dysregulated glycogen synthase kinase-3 (GSK3) may contribute to the pathophysiology of mood disorders and other diseases, and appears to be a target of certain therapeutic drugs. The growing recognition of heightened vulnerability during development to many psychiatric diseases, including mood disorders, led us to test if there are developmental changes in mouse brain GSK3 and its regulation by phosphorylation and by therapeutic drugs. Methods GSK3 levels and phosphorylation were measured at seven ages of development in mouse cerebral cortex and hippocampus. Results Two periods of rapid transitions in GSK3 levels were identified, a large rise between postnatal day 1 and two to three weeks of age, where GSK3 levels were as high as four-fold adult mouse brain levels, and a rapid decline between two to four and eight weeks of age, when adult levels were reached. Inhibitory serine-phosphorylation of GSK3, particularly GSK3β, was extremely high in one-day postnatal mouse brain, and rapidly declined thereafter. These developmental changes in GSK3 were equivalent in male and female cerebral cortex, and differed from other signaling kinases, including Akt, ERK1/2, JNK, and p38 levels and phosphorylation. In contrast to adult mouse brain, where administration of lithium or fluoxetine rapidly and robustly increased serine-phosphorylation of GSK3, in young mice these responses were blunted or absent. Conclusions High brain levels of GSK3 and large fluctuations in its levels and phosphorylation in juvenile and adolescent mouse brain raise the possibility that they may contribute to destabilized mood regulation induced by environmental and genetic factors. PMID:23167932

  1. Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes.

    Science.gov (United States)

    González-Thuillier, Irene; Venegas-Calerón, Mónica; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

    2015-01-01

    Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of β-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

  2. Phosphorylation of sites 3 and 2 in rabbit skeletal muscle glycogen synthase by a multifunctional protein kinase (ATP-citrate lyase kinase)

    International Nuclear Information System (INIS)

    Sheorain, V.S.; Ramakrishna, S.; Benjamin, W.B.; Soderling, T.R.

    1985-01-01

    A multifunctional protein kinase, purified from rat liver as ATP-citrate lyase kinase, has been identified as a glycogen synthase kinase. This kinase catalyzed incorporation of up to 1.5 mol of and]2number 2 PO 4 /mol of synthase subunit associated with a decrease in the glycogen synthase activity ratio from 0.85 to a value of 0.15. Approximately 65-70% of the 34 PO 4 was incorporated into site 3 and 30-35% into site 2 as determined by reverse phase high performance liquid chromatography. This multifunctional kinase was distinguished from glycogen synthase kinase-3 on the basis of nucleotide and protein substrate specificities. Since the phosphate contents in glycogen synthase of sites 3 and 2 are altered in diabetes and by insulin administration, the possible involvement of the multifunctional kinase was explored. Glycogen synthase purified from diabetic rabbits was phosphorylated in vitro by this multifunctional kinase at only 10% of the rate compared to synthase purified from control rabbits. Treatment of the diabetics with insulin restored the synthase to a form that was readily phosphorylated in vitro

  3. Double-Mutated 5-Enol Pyruvylshikimate-3-phosphate Synthase Protein Expressed in MZHG0JG Corn (Zea mays L.) Has No Impact on Toxicological Safety and Nutritional Composition.

    Science.gov (United States)

    Matthews, Bethany A; Launis, Karen L; Bauman, Patricia A; Juba, Nicole C

    2017-09-27

    MZHG0JG corn will offer growers the flexibility to alternate between herbicides with two different modes of action in their weed-management programs, helping to mitigate and manage the evolution of herbicide resistance in weed populations. The proteins conferring herbicide tolerence in MZHG0JG corn, double-mutated 5-enol pyruvylshikimate-3-phosphate synthase protein (mEPSPS) and phosphinothricin acetyltransferase (PAT), as well as the MZHG0JG corn event, have been assessed by regulatory authorities globally and have been determined to be safe for humans, animals, and the environment. In addition to the safety data available for these proteins, further studies were conducted on MZHG0JG corn to assess levels of mEPSPS as compared to previously registered genetically modified (GM) corn. The results support the conclusion of no impact on toxicological safety or nutritional composition.

  4. β-trace protein (prostaglandin D synthase - a stable and reliable protein in perilymph

    Directory of Open Access Journals (Sweden)

    Nekic, Marko

    2005-06-01

    Full Text Available Objective: Beta-trace protein (β-TP has been analysed in human cerebrospinal fluid (CSF and other body fluids. Beta-trace protein is a very sensitive and specific clinical marker and can confirm reliably the presence of CSF in patients with a suspected CSF leakage. Design: Perilymph specimens from the scala vestibuli (n=10 and from the lateral semicircular canal (n=4 were taken from patients undergoing stapedotomy or surgery for acoustic neuroma. During post-mortem examinations perilymph specimens from the scala vestibuli (n=70, the scala tympani (n=11, endolymph specimens (n=21 and CSF specimens (n=17 were obtained. All specimens were analyzed by a one-dimensional immunoelectrophoresis using a polyclonal, monospecific antibody. Results: Specimens from live surgery showed a mean concentration of 51.5 ± 48.9 mg/l β-TP in scala vestibuli perilymph. Specimens from post-mortem examinations revealed a mean concentration of 49.1 ± 17.7 mg/l in CSF, 71.9 ± 29.3 mg/l in perilymph and 68.0 ± 21.7 mg/l in endolymph. There was no evidence of a circadian alteration of β-TP in CSF or inner ear fluids. Conclusions: Our results demonstrated clearly that β-TP is contained in human perilymph and endolymph. This is the first published data that point out the aptitude of the β-TP-test in verifying traces of perilymph, a valuable diagnostic tool for the existence of perilymphatic leaks.

  5. Cloning, expression, and characterization of recombinant nitric oxide synthase-like protein from Bacillus anthracis

    International Nuclear Information System (INIS)

    Midha, Shuchi; Mishra, Rajeev; Aziz, M.A.; Sharma, Meenakshi; Mishra, Ashish; Khandelwal, Puneet; Bhatnagar, Rakesh

    2005-01-01

    Nitric oxide synthase (NOS) is amongst a family of evolutionarily conserved enzymes, involved in a multi-turnover process that results in NO as a product. The significant role of NO in various pathological and physiological processes has created an interest in this enzyme from several perspectives. This study describes for the first time, cloning and expression of a NOS-like protein, baNOS, from Bacillus anthracis, a pathogenic bacterium responsible for causing anthrax. baNOS was expressed in Escherichia coli as a soluble and catalytically active enzyme. Homology models generated for baNOS indicated that the key structural features that are involved in the substrate and active site interaction have been highly conserved. Further, the behavior of baNOS in terms of heme-substrate interactions and heme-transitions was studied in detail. The optical perturbation spectra of the heme domain demonstrated that the ligands perturb the heme site in a ligand specific manner. baNOS forms a five-coordinate, high-spin complex with L-arginine analogs and a six-coordinate low-spin complex with inhibitor imidazole. Studies indicated that the binding of L-arginine, N ω -hydroxy-L-arginine, and imidazole produces various spectroscopic species that closely correspond to the equivalent complexes of mammalian NOS. The values of spectral binding constants further corroborated these results. The overall conservation of the key structural features and the correlation of heme-substrate interactions in baNOS and mammalian NOS, thus, point towards an interesting phenomenon of convergent evolution. Importantly, the NO generated by NOS of mammalian macrophages plays a potent role in antimicrobicidal activity. Because of the existence of high structural and behavioral similarity between mammalian NOS and baNOS, we propose that NO produced by B. anthracis may also have a pivotal pathophysiological role in anthrax infection. Therefore, this first report of characterization of a NOS-like protein

  6. Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis.

    Science.gov (United States)

    Lardizabal, K D; Metz, J G; Sakamoto, T; Hutton, W C; Pollard, M R; Lassner, M W

    2000-03-01

    Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.

  7. Kernel based machine learning algorithm for the efficient prediction of type III polyketide synthase family of proteins

    Directory of Open Access Journals (Sweden)

    Mallika V

    2010-03-01

    Full Text Available Type III Polyketide synthases (PKS are family of proteins considered to have significant role in the biosynthesis of various polyketides in plants, fungi and bacteria. As these proteins show positive effects to human health, more researches are going on regarding this particular protein. Developing a tool to identify the probability of sequence, being a type III polyketide synthase will minimize the time consumption and manpower efforts. In this approach, we have designed and implemented PKSIIIpred, a high performance prediction server for type III PKS where the classifier is Support Vector Machine (SVM. Based on the limited training dataset, the tool efficiently predicts the type III PKS superfamily of proteins with high sensitivity and specificity. PKSIIIpred is available at http://type3pks.in/prediction/. We expect that this tool may serve as a useful resource for type III PKS researchers. Currently work is being progressed for further betterment of prediction accuracy by including more sequence features in the training dataset.

  8. Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis.

    Science.gov (United States)

    Zhang, Chunyu; Zhang, Wei; Ren, Guodong; Li, Delin; Cahoon, Rebecca E; Chen, Ming; Zhou, Yongming; Yu, Bin; Cahoon, Edgar B

    2015-08-01

    Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis. © 2015 American Society of

  9. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    International Nuclear Information System (INIS)

    Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

    1986-01-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca 2+ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting 32 P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32 P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor

  10. Pu-erh Tea Reduces Nitric Oxide Levels in Rats by Inhibiting Inducible Nitric Oxide Synthase Expression through Toll-Like Receptor 4

    Science.gov (United States)

    Xu, Yang; Wang, Guan; Li, Chunjie; Zhang, Min; Zhao, Hang; Sheng, Jun; Shi, Wei

    2012-01-01

    Pu-erh tea undergoes a unique fermentation process and contains theabrownins, polysaccharides and caffeine; although it is unclear about which component is associated with the down regulation of nitric oxide levels or how this process is mediated. To address this question we examined the effects of pu-erh tea on nitric oxide synthase (NOS) genes. Cohorts of rats were separately given four-week treatments of water as control, pu-erh tea, or the tea components: theabrownins, caffeine or polysaccharides. Five experimental groups were injected with lipopolysaccharides (LPS) to induce nitric oxide (NO) production, while the corresponding five control groups were injected with saline as a negative control. The serum and liver NO concentrations were examined and the NOS expression of both mRNA and protein was measured in liver. The results showed that the rats which were fed pu-erh tea or polysaccharides had lower levels of NO which corresponded with the down-regulation of inducible nitric oxide synthase (iNOS) expression. We further demonstrate that this effect is mediated through reduction of Toll-like receptor 4 (TLR4) signaling. Thus we find that the polysaccharide components in pu-erh tea reduce NO levels in an animal model by inhibiting the iNOS expression via signaling through TLR4. PMID:22837686

  11. of endothelial nitric oxide synthase gene and serum level of vascular ...

    African Journals Online (AJOL)

    uwerhiavwe

    Davignon and Ganz, 2004). NO is synthe- sized via a reaction that includes the conversion of L- arginine to L-citruline catalyzed by endothelial nitric oxide synthase (eNOS), which is one of the three isoforms of the enzyme (Mayer and Hemmens, 1997) ...

  12. Aldosterone-Synthase Gene Polymorphism is Associated with Blood Pressure Levels and Left Ventricle Mass Index

    Czech Academy of Sciences Publication Activity Database

    Horký, K.; Jáchymová, M.; Heller, S.; Linhart, A.; Hlubocká, Z.; Umnerová, V.; Peleška, Jan; Pavlíková, Markéta; Jindra, A.

    2004-01-01

    Roč. 204, 1 suppl. (2004), s. 35 ISSN 0014-2565. [World Congress of Internal Medicine /27./. 26.09.2004-01.10.2004, Granada] R&D Projects: GA MŠk LN00B107 Keywords : aldosterone synthase (CYP11B) * genetic polymorphism * arterial hypertension * left ventricular hypertrophy Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery

  13. Exploring Protein Interactions on a Minimal Type II Polyketide Synthase Using a Yeast Two-Hybrid System

    Directory of Open Access Journals (Sweden)

    Gaetano Castaldo

    2005-01-01

    Full Text Available Interactions between proteins that form the ’minimal’ type II polyketide synthase in the doxorubicin producing biosynthetic pathway from Streptomyces peucetius were investigated using a yeast two-hybrid system (Y2H. Proteins that function as the so called ’chain length factor’ (DpsB and putative transacylase (DpsD were found to interact with the ketosynthase subunit (DpsA, which can also interact with itself. On the basis of these results we propose a head-to-tail homodimeric structure, which is consistent with previously published in vivo mutagenesis studies. No interactions were found between the acyl-carrier protein (DpsG and any of the other constituents of the complex, however, transient interactions, not detectable using the Y2H system, cannot be discounted and warrant further investigation.

  14. Evolutionary history of callose synthases in terrestrial plants with emphasis on proteins involved in male gametophyte development.

    Directory of Open Access Journals (Sweden)

    Lenka Záveská Drábková

    Full Text Available Callose is a plant-specific polysaccharide (β-1,3-glucan playing an important role in angiosperms in many developmental processes and responses to biotic and abiotic stresses. Callose is synthesised at the plasma membrane of plant cells by callose synthase (CalS and, among others, represents the main polysaccharide in the callose wall surrounding the tetrads of developing microspores and in the growing pollen tube wall. CalS proteins involvement in spore development is a plesiomorphic feature of terrestrial plants, but very little is known about their evolutionary origin and relationships amongst the members of this protein family. We performed thorough comparative analyses of callose synthase family proteins from major plant lineages to determine their evolutionary history across the plant kingdom. A total of 1211 candidate CalS sequences were identified and compared amongst diverse taxonomic groups of plants, from bryophytes to angiosperms. Phylogenetic analyses identified six main clades of CalS proteins and suggested duplications during the evolution of specialised functions. Twelve family members had previously been identified in Arabidopsis thaliana. We focused on five CalS subfamilies directly linked to pollen function and found that proteins expressed in pollen evolved twice. CalS9/10 and CalS11/12 formed well-defined clades, whereas pollen-specific CalS5 was found within subfamilies that mostly did not express in mature pollen vegetative cell, although were found in sperm cells. Expression of five out of seven mature pollen-expressed CalS genes was affected by mutations in bzip transcription factors. Only three subfamilies, CalS5, CalS10, and CalS11, however, formed monophyletic, mostly conserved clades. The pairs CalS9/CalS10, CalS11/CalS12 and CalS3 may have diverged after angiosperms diversified from lycophytes and bryophytes. Our analysis of fully sequenced plant proteins identified new evolutionary lineages of callose synthase

  15. Skeletal muscle neuronal nitric oxide synthase micro protein is reduced in people with impaired glucose homeostasis and is not normalized by exercise training.

    Science.gov (United States)

    Bradley, Scott J; Kingwell, Bronwyn A; Canny, Benedict J; McConell, Glenn K

    2007-10-01

    Skeletal muscle inducible nitric oxide synthase (NOS) protein is greatly elevated in people with type 2 diabetes mellitus, whereas endothelial NOS is at normal levels. Diabetic rat studies suggest that skeletal muscle neuronal NOS (nNOS) micro protein expression may be reduced in human insulin resistance. The aim of this study was to determine whether skeletal muscle nNOSmicro protein expression is reduced in people with impaired glucose homeostasis and whether exercise training increases nNOSmicro protein expression in these individuals because exercise training increases skeletal muscle nNOSmicro protein in rats. Seven people with type 2 diabetes mellitus or prediabetes (impaired fasting glucose and/or impaired glucose tolerance) and 7 matched (sex, age, fitness, body mass index, blood pressure, lipid profile) healthy controls aged 36 to 60 years participated in this study. Vastus lateralis muscle biopsies for nNOSmicro protein determination were obtained, aerobic fitness was measured (peak pulmonary oxygen uptake [Vo(2) peak]), and glucose tolerance and insulin homeostasis were assessed before and after 1 and 4 weeks of cycling exercise training (60% Vo(2) peak, 50 minutes x 5 d wk(-1)). Skeletal muscle nNOSmicro protein was significantly lower (by 32%) in subjects with type 2 diabetes mellitus or prediabetes compared with that in controls before training (17.7 +/- 1.2 vs 26.2 +/- 3.4 arbitrary units, P glucose homeostasis have reduced skeletal muscle nNOSmicro protein content. However, because exercise training improves insulin sensitivity without influencing skeletal muscle nNOSmicro protein expression, it seems that changes in skeletal muscle nNOSmicro protein are not central to the control of insulin sensitivity in humans and therefore may be a consequence rather than a cause of diabetes.

  16. Homology analyses of the protein sequences of fatty acid synthases from chicken liver, rat mammary gland, and yeast

    International Nuclear Information System (INIS)

    Chang, Soo-Ik; Hammes, G.G.

    1989-01-01

    Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chicken and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the β-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution

  17. Glycogen Synthase Kinase 3 Inactivation Induces Cell Senescence through Sterol Regulatory Element Binding Protein 1-Mediated Lipogenesis in Chang Cells.

    Science.gov (United States)

    Kim, You-Mie; Song, Insun; Seo, Yong-Hak; Yoon, Gyesoon

    2013-12-01

    Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence. We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 µM) of deferoxamine (DFO) and H2O2. In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3α (GSK3α) and β corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3α and β also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence. GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.

  18. A Cyanobacterial Chlorophyll Synthase-HliD Complex Associates with the Ycf39 Protein and the YidC/Alb3 Insertase[W][OPEN

    Science.gov (United States)

    Chidgey, Jack W.; Linhartová, Markéta; Komenda, Josef; Jackson, Philip J.; Dickman, Mark J.; Canniffe, Daniel P.; Koník, Peter; Pilný, Jan; Hunter, C. Neil; Sobotka, Roman

    2014-01-01

    Macromolecular membrane assemblies of chlorophyll-protein complexes efficiently harvest and trap light energy for photosynthesis. To investigate the delivery of chlorophylls to the newly synthesized photosystem apoproteins, a terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), was tagged in the cyanobacterium Synechocystis PCC 6803 (Synechocystis) and used as bait in pull-down experiments. We retrieved an enzymatically active complex comprising ChlG and the high-light-inducible protein HliD, which associates with the Ycf39 protein, a putative assembly factor for photosystem II, and with the YidC/Alb3 insertase. 2D electrophoresis and immunoblotting also provided evidence for the presence of SecY and ribosome subunits. The isolated complex contained chlorophyll, chlorophyllide, and carotenoid pigments. Deletion of hliD elevated the level of the ChlG substrate, chlorophyllide, more than 6-fold; HliD is apparently required for assembly of FLAG-ChlG into larger complexes with other proteins such as Ycf39. These data reveal a link between chlorophyll biosynthesis and the Sec/YidC-dependent cotranslational insertion of nascent photosystem polypeptides into membranes. We expect that this close physical linkage coordinates the arrival of pigments and nascent apoproteins to produce photosynthetic pigment-protein complexes with minimal risk of accumulating phototoxic unbound chlorophylls. PMID:24681617

  19. Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer

    International Nuclear Information System (INIS)

    Kaur, Jatinder; Sawhney, Meenakshi; DattaGupta, Siddartha; Shukla, Nootan K; Srivastava, Anurag; Ralhan, Ranju

    2010-01-01

    We reported increased levels of Phosphatidyl Inositol synthase (PI synthase), (enzyme that catalyses phosphatidyl inositol (PI) synthesis-implicated in intracellular signaling and regulation of cell growth) in smokeless tobacco (ST) exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC) and premalignant lesions (leukoplakia), and identified the downstream signaling proteins in PI synthase pathway that are perturbed by smokeless tobacco (ST) exposure. Tissue microarray (TMA) Immunohistochemistry, Western blotting, Confocal laser scan microscopy, RT-PCR were performed to define the expression of PI synthase in clinical samples and in oral cell culture systems. Significant increase in PI synthase immunoreactivity was observed in premalignant lesions and OSCCs as compared to oral normal tissues (p = 0.000). Further, PI synthase expression was significantly associated with de-differentiation of OSCCs, (p = 0.005) and tobacco consumption (p = 0.03, OR = 9.0). Exposure of oral cell systems to smokeless tobacco (ST) in vitro confirmed increase in PI synthase, Phosphatidylinositol 3-kinase (PI3K) and cyclin D1 levels. Collectively, increased PI synthase expression was found to be an early event in oral cancer and a target for smokeless tobacco

  20. A Ser/Thr protein kinase phosphorylates MA-ACS1 (Musa acuminata 1-aminocyclopropane-1-carboxylic acid synthase 1) during banana fruit ripening.

    Science.gov (United States)

    Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

    2012-08-01

    1-Aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the rate-limiting step in ethylene biosynthesis during ripening. ACS isozymes are regulated both transcriptionally and post-translationally. However, in banana, an important climacteric fruit, little is known about post-translational regulation of ACS. Here, we report the post-translational modification of MA-ACS1 (Musa acuminata ACS1), a ripening inducible isozyme in the ACS family, which plays a key role in ethylene biosynthesis during banana fruit ripening. Immunoprecipitation analyses of phospholabeled protein extracts from banana fruit using affinity-purified anti-MA-ACS1 antibody have revealed phosphorylation of MA-ACS1, particularly in ripe fruit tissue. We have identified the induction of a 41-kDa protein kinase activity in pulp at the onset of ripening. The 41-kDa protein kinase has been identified as a putative protein kinase by MALDI-TOF/MS analysis. Biochemical analyses using partially purified protein kinase fraction from banana fruit have identified the protein kinase as a Ser/Thr family of protein kinase and its possible involvement in MA-ACS1 phosphorylation during ripening. In vitro phosphorylation analyses using synthetic peptides and site-directed mutagenized recombinant MA-ACS1 have revealed that serine 476 and 479 residues at the C-terminal region of MA-ACS1 are phosphorylated. Overall, this study provides important novel evidence for in vivo phosphorylation of MA-ACS1 at the molecular level as a possible mechanism of post-translational regulation of this key regulatory protein in ethylene signaling pathway in banana fruit during ripening.

  1. A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

    Science.gov (United States)

    Du, Z Q; Wang, J Y

    2015-10-27

    Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins.

  2. The role of ß-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower

    DEFF Research Database (Denmark)

    González-Mellado, Damián; von Wettstein, Penny; Garcés, Rafael

    2010-01-01

    The ß-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing...... seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous...... proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons...

  3. Expression analysis of cellulose synthase and main cytoskeletal protein genes in flax (Linum usitatissimum L.).

    Science.gov (United States)

    Galinousky, Dmitry; Padvitski, Tsimafei; Bayer, Galina; Pirko, Yaroslav; Pydiura, Nikolay; Anisimova, Natallia; Nikitinskaya, Tatyana; Khotyleva, Liubov; Yemets, Alla; Kilchevsky, Aleksandr; Blume, Yaroslav

    2017-08-09

    Fiber flax is an important source of natural fiber and a comprehensive model for the plant fiber biogenesis studies. Cellulose-synthase (CesA) and cytoskeletal genes are known to be important for the cell wall biogenesis in general and for the biogenesis of flax fibers in particular. Currently, knowledge about activity of these genes during the plant growth is limited. In this study, we have investigated flax fiber biogenesis by measuring expression of CesA and cytoskeletal genes at two stages of the flax development (seedlings and stems at the rapid growth stage) in several flax subspecies (elongatum, mediterraneum, crepitans). RT-qPCR has been used to quantify the expression of LusСesA1, LusСesA4, LusСesA7, LusСesA6, Actin, and α-Tubulin genes in plant samples. We report that CesA genes responsible for the secondary cell wall synthesis (LusCesA4, LusCesA7) have different expression pattern compared with CesA genes responsible for the primary cell wall synthesis (LusCesA1, LusCesA6): an average expression of LusCesA4 and LusCesA7 genes is relatively high in seedlings and further increases in stems at the rapid growth stage, whereas an average expression of LusCesA1 and LusCesA6 genes decreases. Interestingly, LusCesA1 is the only studied gene with different expression dynamics between the flax subspecies: its expression decreases by 5.2-10.7 folds in elongatum and mediterraneum but does not change in crepitans subspecies when the rapid growth stage and seedlings are compared. The expression of cytoskeleton genes (coding actin and α-tubulin) is relatively stable and significantly higher than the expression of cellulose-synthase genes in all the studied samples. © 2017 International Federation for Cell Biology.

  4. The alpha2-5'AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading

    DEFF Research Database (Denmark)

    Jørgensen, Sebastian B; Nielsen, Jakob N.; Birk, Jesper Bratz

    2004-01-01

    The 5'AMP-activated protein kinase (AMPK) is a potential antidiabetic drug target. Here we show that the pharmacological activation of AMPK by 5-aminoimidazole-1-beta-4-carboxamide ribofuranoside (AICAR) leads to inactivation of glycogen synthase (GS) and phosphorylation of GS at Ser 7 (site 2). ...

  5. Effects of inhibitors of protein kinase C and NO-synthase on the radiation-induced cytogenetic adaptive response in Chinese hamster cells in culture

    International Nuclear Information System (INIS)

    Gil'yano, N.Ya.; Bondarev, G.N.; Bikineeva, E.G.; Krasotskaya, G.I.; Noskin, L.A.

    2001-01-01

    The effect of the serine-threonin kinase inhibitor - staurosporine and inhibitor of NO-synthase - L-NAME on the radiation-induced adaptive response were studied in fibroblasts of Chinese hamster in culture. It is shown that staurosporine and L-NAME inhibit cytogenetic adaptive response induced by β-particles in low doses. Inhibition is not connected with radiosensitizing effect of these agents. L-NAME decreases significantly the γ-rays-induced chromosome aberration yield also. Study confirms the role of protein kinase C in induction of the adaptive response and participation of NO-synthase in this process is noticed for the first time [ru

  6. Molecular cloning and characterization of two β-ketoacyl-acyl carrier protein synthase I genes from Jatropha curcas L.

    Science.gov (United States)

    Xiong, Wangdan; Wei, Qian; Wu, Pingzhi; Zhang, Sheng; Li, Jun; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

    2017-07-01

    The β-ketoacyl-acyl carrier protein synthase I (KASI) is involved in de novo fatty acid biosynthesis in many organisms. Two putative KASI genes, JcKASI-1 and JcKASI-2, were isolated from Jatropha curcas. The deduced amino acid sequences of JcKASI-1 and JcKASI-2 exhibit around 83.8% and 72.5% sequence identities with AtKASI, respectively, and both contain conserved Cys-His-Lys-His-Phe catalytic active sites. Phylogenetic analysis indicated that JcKASI-2 belongs to a clade with several KASI proteins from dicotyledonous plants. Both JcKASI genes were expressed in multiple tissues, most strongly in filling stage seeds of J. curcas. Additionally, the JcKASI-1 and JcKASI-2 proteins were both localized to the plastids. Expressing JcKASI-1 in the Arabidopsis kasI mutant rescued the mutant's phenotype and restored the fatty acid composition and oil content in seeds to wild-type, but expressing JcKASI-2 in the Arabidopsis kasI mutant resulted in only partial rescue. This implies that JcKASI-1 and JcKASI-2 exhibit partial functional redundancy and KASI genes play a universal role in regulating fatty acid biosynthesis, growth, and development in plants. Copyright © 2017 Elsevier GmbH. All rights reserved.

  7. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China); Zhang, Qunye, E-mail: wz.zhangqy@sdu.edu.cn [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Ministry of Public Health, Qilu Hospital, Shandong University, Jinan, Shandong (China); Li, Guorong, E-mail: grli@sdnu.edu.cn [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China)

    2015-03-13

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.

  8. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    International Nuclear Information System (INIS)

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi; Zhang, Qunye; Li, Guorong

    2015-01-01

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation

  9. Regulatory role of tumor necrosis factor receptor-associated factor 6 in breast cancer by activating the protein kinase B/glycogen synthase kinase 3β signaling pathway.

    Science.gov (United States)

    Shen, Hongyu; Li, Liangpeng; Yang, Sujin; Wang, Dandan; Zhou, Siying; Chen, Xiu; Tang, Jinhai

    2017-08-01

    Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an endogenous adaptor of innate and adaptive immune responses, and serves a crucial role in tumor necrosis factor receptor and toll‑like/interleukin‑1 receptor signaling. Although studies have demonstrated that TRAF6 has oncogenic activity, its potential contributions to breast cancer in human remains largely uninvestigated. The present study examined the expression levels and function of TRAF6 in breast carcinoma (n=32) and adjacent healthy (n=25) tissue samples. Compared with adjacent healthy tissues, TRAF6 protein expression levels were significantly upregulated in breast cancer tissues. Reverse transcription‑quantitative polymerase chain reaction analysis revealed a significant upregulation of the cellular proliferative marker Ki‑67 and proliferation cell nuclear antigen expression levels in breast carcinoma specimens. Furthermore, protein expression levels of the accessory molecule, transforming growth factor β‑activated kinase 1 (TAK1), were significantly increased in breast cancer patients, as detected by western blot analysis. As determined by MTT assay, TRAF6 exerted profoundly proliferative effects in the MCF‑7 breast cancer cell line; however, these detrimental effects were ameliorated by TAK1 inhibition. Notably, protein kinase B (AKT)/glycogen synthase kinase (GSK)3β phosphorylation levels were markedly upregulated in breast cancer samples, compared with adjacent healthy tissues. In conclusion, an altered TRAF6‑TAK1 axis and its corresponding downstream AKT/GSK3β signaling molecules may contribute to breast cancer progression. Therefore, TRAF6 may represent a potential therapeutic target for the treatment of breast cancer.

  10. Prostaglandin E synthase interacts with inducible heat shock protein 70 after heat stress in bovine primary dermal fibroblast cells.

    Science.gov (United States)

    Richter, Constanze; Viergutz, Torsten; Schwerin, Manfred; Weitzel, Joachim M

    2015-01-01

    Exposure to heat stress in dairy cows leads to undesired side effects that are reflected by complex alterations in endocrine parameters, such as reduced progesterone, estradiol, and thyroid hormone concentrations. These endocrine maladaptation leads to failure to resume cyclicity, a poor uterine environment and inappropriate immune responses in postpartum dairy cows. Prostaglandins (PG's) are lipid mediators, which serve as signal molecules in response to various external stimuli as well as to cell-specific internal signal molecules. A central role in PG synthesis plays prostaglandin E synthase (PGES) that catalyzes the isomerization of PGH2 to PGE2 .The present study was conducted to investigate heat stress associated PGES expression. Expression of PGES and inducible heat shock protein 70 (HSP70), as a putative chaperonic protein, was studied in bovine primary fibroblasts under different heat shock conditions. Bovine primary fibroblasts produce PGE2 at homoiothermical norm temperature (38.5°C in bovine), but reduce PGE2 production rates under extreme heat stress (at 45°C for 6 h). By contrast, PGE2 production rates are maintained after a milder heat stress (at 41.5°C for 6 h). PGE2 synthesis is abolished by application of cyclooxygenase inhibitor indomethacin, indicating de novo synthesis. Heat stress increases HSP70 but not PGES protein concentrations. HSP70 physically interacts with PGES and the PGES-HSP70 complex did not dissociate upon heat stress at 45°C even after returning the cells to 37°C. The PGE2 production negatively correlates with the portion of PGES-HSP70 complex. These results suggest a protein interaction between HSP70 and PGES in dermal fibroblast cells. Blockage of PGES protein by HSP70 seems to interfere with the regulatory processes essential for cellular adaptive protection. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

  11. Deciphering the interplay between cysteine synthase and thiol cascade proteins in modulating Amphotericin B resistance and survival of Leishmania donovani under oxidative stress

    Directory of Open Access Journals (Sweden)

    Kuljit Singh

    2017-08-01

    Full Text Available Leishmania donovani is the causative organism of the neglected human disease known as visceral leishmaniasis which is often fatal, if left untreated. The cysteine biosynthesis pathway of Leishmania may serve as a potential drug target because it is different from human host and regulates downstream components of redox metabolism of the parasites; essential for their survival, pathogenicity and drug resistance. However, despite the apparent dependency of redox metabolism of cysteine biosynthesis pathway, the role of L. donovani cysteine synthase (LdCS in drug resistance and redox homeostasis has been unexplored. Herein, we report that over-expression of LdCS in Amphotericin B (Amp B sensitive strain (S1-OE modulates resistance towards oxidative stress and drug pressure. We observed that antioxidant enzyme activities were up-regulated in S1-OE parasites and these parasites alleviate intracellular reactive oxygen species (ROS efficiently by maintaining the reduced thiol pool. In contrast to S1-OE parasites, Amp B sensitive strain (S1 showed higher levels of ROS which was positively correlated with the protein carbonylation levels and negatively correlated with cell viability. Moreover, further investigations showed that LdCS over-expression also augments the ROS-primed induction of LdCS-GFP as well as endogenous LdCS and thiol pathway proteins (LdTryS, LdTryR and LdcTXN in L. donovani parasites; which probably aids in stress tolerance and drug resistance. In addition, the expression of LdCS was found to be up-regulated in Amp B resistant isolates and during infective stationary stages of growth and consistent with these observations, our ex vivo infectivity studies confirmed that LdCS over-expression enhances the infectivity of L. donovani parasites. Our results reveal a novel crosstalk between LdCS and thiol metabolic pathway proteins and demonstrate the crucial role of LdCS in drug resistance and redox homeostasis of Leishmania. Keywords

  12. The Influence of Hyperoxia On Heat Shock Proteins Expression and Nitric Oxide Synthase Activity – the Review

    Directory of Open Access Journals (Sweden)

    Szyller Jakub

    2017-03-01

    Full Text Available Any stay in an environment with an increased oxygen content (a higher oxygen partial pressure, pO2 and an increased pressure (hyperbaric conditions leads to an intensification of oxidative stress. Reactive oxygen species (ROS damage the molecules of proteins, nucleic acids, cause lipid oxidation and are engaged in the development of numerous diseases, including diseases of the circulatory system, neurodegenerative diseases, etc. There are certain mechanisms of protection against unfavourable effects of oxidative stress. Enzymatic and non-enzymatic systems belong to them. The latter include, among others, heat shock proteins (HSP. Their precise role and mechanism of action have been a subject of intensive research conducted in recent years. Hyperoxia and hyperbaria also have an effect on the expression and activity of nitrogen oxide synthase (NOS. Its product - nitrogen oxide (NO can react with reactive oxygen species and contribute to the development of nitrosative stress. NOS occurs as isoforms in various tissues and exhibit different reactions to the discussed factors. The authors have prepared a brief review of research determining the effect of hyperoxia and hyperbaria on HSP expression and NOS activity.

  13. The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesin

    Directory of Open Access Journals (Sweden)

    Pereira Maristela

    2009-12-01

    Full Text Available Abstract Background The pathogenic fungus Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM. This is a pulmonary mycosis acquired by inhalation of fungal airborne propagules that can disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside the host, P. brasiliensis may use the glyoxylate cycle for intracellular survival. Results Here, we provide evidence that the malate synthase of P. brasiliensis (PbMLS is located on the fungal cell surface, and is secreted. PbMLS was overexpressed in Escherichia coli, and polyclonal antibody was obtained against this protein. By using Confocal Laser Scanning Microscopy, PbMLS was detected in the cytoplasm and in the cell wall of the mother, but mainly of budding cells of the P. brasiliensis yeast phase. PbMLSr and its respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis with in vitro cultured epithelial cells A549. Conclusion These observations indicated that cell wall-associated PbMLS could be mediating the binding of fungal cells to the host, thus contributing to the adhesion of fungus to host tissues and to the dissemination of infection, behaving as an anchorless adhesin.

  14. Mechanical unloading of the failing human heart fails to activate the protein kinase B/Akt/glycogen synthase kinase-3beta survival pathway.

    Science.gov (United States)

    Razeghi, Peter; Bruckner, Brian A; Sharma, Saumya; Youker, Keith A; Frazier, O H; Taegtmeyer, Heinrich

    2003-01-01

    Left ventricular assist device (LVAD) support of the failing human heart improves myocyte function and increases cell survival. One potential mechanism underlying this phenomenon is activation of the protein kinase B (PKB)/Akt/glycogen synthase kinase-3beta (GSK-3beta) survival pathway. Left ventricular tissue was obtained both at the time of implantation and explantation of the LVAD (n = 11). Six patients were diagnosed with idiopathic dilated cardiomyopathy, 4 patients with ischemic cardiomyopathy and 1 patient with peripartum cardiomyopathy. The mean duration of LVAD support was 205 +/- 35 days. Myocyte diameter and phosphorylation of ERK were used as indices for reverse remodeling. Transcript levels of genes required for the activation of PKB/Akt (insulin-like growth factor-1, insulin receptor substrate-1) were measured by quantitative RT-PCR. In addition, we measured the relative activity of PKB/Akt and GSK-3beta, and assayed for molecular and histological indices of PKB/Akt activation (cyclooxygenase mRNA levels and glycogen levels). Myocyte diameter and phosphorylation of ERK decreased with LVAD support. In contrast, none of the components of the PKB/Akt/GSK-3beta pathway changed significantly with mechanical unloading. The PKB/Akt/GSK-3beta pathway is not activated during LVAD support. Other signaling pathways must be responsible for the improvement of cellular function and cell survival during LVAD support. Copyright 2003 S. Karger AG, Basel

  15. Quantitation of secretory protein levels by radioimmunoassay

    International Nuclear Information System (INIS)

    Klein, J.L.; Dawson, J.R.

    1978-01-01

    A radioimmunoassay was designed for the detection of secretory protein, a component of secretory immunoglobulin A, in human serum. The assay uses free secretory protein isolated from human colostrum, and antisera raised in rabbits to be purified antigen. The mean level of secretory protein in the control group was 2.34+-0.41 μg/ml (mean+-S.E.M.). The level in cord blood was slightly lower (0.74+-0.26 μg/ml), while the level in patients with ovarian carcinoma was significantly increased (12.67+-1.43 μg/ml). Pregnant women have increasingly secretory protein levels with increasing length of gestation (5.86+-2.02, 11.55+-1.30 and 17.00+-1.16 μg/ml for the first, second and third trimesters, respectively. (Auth.)

  16. Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation.

    Science.gov (United States)

    Kobayashi, Soushi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

    2015-07-01

    Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca(2+)-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca(2+)-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca(2+). These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. The structure of Rauvolfia serpentina strictosidine synthase is a novel six-bladed beta-propeller fold in plant proteins.

    Science.gov (United States)

    Ma, Xueyan; Panjikar, Santosh; Koepke, Juergen; Loris, Elke; Stöckigt, Joachim

    2006-04-01

    The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of approximately 2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler-type reaction and represents a novel six-bladed beta-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed beta-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family.

  18. Sterol regulatory element-binding protein-1 participates in the regulation of fatty acid synthase expression in colorectal neoplasia.

    Science.gov (United States)

    Li, J N; Mahmoud, M A; Han, W F; Ripple, M; Pizer, E S

    2000-11-25

    Endogenous fatty acid synthesis has been observed in certain rapidly proliferating normal and neoplastic tissues. Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate the expression of lipogenic genes including fatty acid synthase (FAS), the major biosynthetic enzyme for fatty acid synthesis. We have previously shown that SREBP-1, FAS, and Ki-67, a proliferation marker, colocalized in the crypts of the fetal gastrointestinal tract epithelium. This study sought to determine whether SREBP-1 participates in the regulation of proliferation-associated fatty acid synthesis in colorectal neoplasia. An immunohistochemical analysis of SREBP-1, FAS, and Ki-67 expression in 25 primary human colorectal carcinoma specimens showed colocalization in 22 of these. To elucidate a functional linkage between SREBP-1 activation and proliferation-associated FA synthesis, SREBP-1 and FAS content were assayed during the adaptive response of cultured HCT116 colon carcinoma cells to pharmacological inhibition of FA synthesis. Cerulenin and TOFA each inhibited the endogenous synthesis of fatty acids in a dose-dependent manner and each induced increases in both precursor and mature forms of SREBP-1. Subsequently, both the transcriptional activity of the FAS promoter in a luciferase reporter gene construct and the FAS expression increased. These results demonstrate that tumor cells recognize and respond to a deficiency in endogenous fatty acid synthesis by upregulating both SREBP-1 and FAS expression and support the model that SREBP-1 participates in the transcriptional regulation of lipogenic genes in colorectal neoplasia. Copyright 2000 Academic Press.

  19. Sphingomyelin synthase-related protein SMSr is a suppressor of ceramide-induced mitochondrial apoptosis

    DEFF Research Database (Denmark)

    Tafesse, Fikadu G.; Vacaru, Ana M.; Bosma, Elleke Fenna

    2014-01-01

    ceramide-induced cell death and that SMSr-mediated ceramide homeostasis requires the N-terminal sterile a-motif, or SAM domain, of the enzyme. These results define ER ceramides as bona fide transducers of mitochondrial apoptosis and indicate a primary role of SMSr in monitoring ER ceramide levels...

  20. Increased expression and local accumulation of the Prion Protein, Alzheimer Aβ peptides, superoxide dismutase 1, and Nitric oxide synthases 1 & 2 in muscle in a rabbit model of diabetes

    Directory of Open Access Journals (Sweden)

    Bitel Claudine L

    2010-09-01

    Full Text Available Abstract Background Muscle disease associated with different etiologies has been shown to produce localized accumulations of amyloid and oxidative stress-related proteins that are more commonly associated with neurodegeneration in the brain. In this study we examined changes in muscle tissue in a classic model of diabetes and hyperglycemia in rabbits to determine if similar dysregulation of Alzheimer Aβ peptides, the prion protein (PrP, and superoxide dismutase 1 (SOD1, as well as nitric oxide synthases is produced in muscle in diabetic animals. This wild-type rabbit model includes systemic physiological expression of human-like Alzheimer precursor proteins and Aβ peptides that are considered key in Alzheimer protein studies. Results Diabetes was produced in rabbits by injection of the toxic glucose analogue alloxan, which selectively enters pancreatic beta cells and irreversibly decreases insulin production, similar to streptozotocin. Quadriceps muscle from rabbits 16 wks after onset of diabetes and hyperglycemia were analyzed with biochemical and in situ methods. Immunoblots of whole muscle protein samples demonstrated increased PrP, SOD1, as well as neuronal and inducible Nitric oxide synthases (NOS1 and NOS2 in diabetic muscle. In contrast, we detected little change in Alzheimer Aβ precursor protein expression, or BACE1 and Presenilin 1 levels. However, Aβ peptides measured by ELISA increased several fold in diabetic muscle, suggesting a key role for Aβ cleavage in muscle similar to Alzheimer neurodegeneration in this diabetes model. Histological changes in diabetic muscle included localized accumulations of PrP, Aβ, NOS1 and 2, and SOD1, and evidence of increased central nuclei and cell infiltration. Conclusions The present study provides evidence that several classic amyloid and oxidative stress-related disease proteins coordinately increase in overall expression and form localized accumulations in diabetic muscle. The present study

  1. Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String Configuration

    KAUST Repository

    Trujillo, Uldaeliz

    2013-02-28

    The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP

  2. Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String Configuration

    KAUST Repository

    Trujillo, Uldaeliz; Vá zquez-Rosa, Edwin; Oyola-Robles, Delise; Stagg, Loren J.; Vassallo, David A.; Vega, Irving E.; Arold, Stefan T.; Baerga-Ortiz, Abel

    2013-01-01

    The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP

  3. Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration.

    Directory of Open Access Journals (Sweden)

    Uldaeliz Trujillo

    Full Text Available The polyunsaturated fatty acid (PUFA synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect and in structural stabilization of the multidomain protein (synergistic effect. While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of

  4. Protein kinase A-dependent Neuronal Nitric Oxide Synthase Activation Mediates the Enhancement of Baroreflex Response by Adrenomedullin in the Nucleus Tractus Solitarii of Rats

    Directory of Open Access Journals (Sweden)

    Ho I-Chun

    2011-05-01

    Full Text Available Abstract Background Adrenomedullin (ADM exerts its biological functions through the receptor-mediated enzymatic mechanisms that involve protein kinase A (PKA, or neuronal nitric oxide synthase (nNOS. We previously demonstrated that the receptor-mediated cAMP/PKA pathway involves in ADM-enhanced baroreceptor reflex (BRR response. It remains unclear whether ADM may enhance BRR response via activation of nNOS-dependent mechanism in the nucleus tractus solitarii (NTS. Methods Intravenous injection of phenylephrine was administered to evoke the BRR before and at 10, 30, and 60 min after microinjection of the test agents into NTS of Sprague-Dawley rats. Western blotting analysis was used to measure the level and phosphorylation of proteins that involved in BRR-enhancing effects of ADM (0.2 pmol in NTS. The colocalization of PKA and nNOS was examined by immunohistochemical staining and observed with a laser confocal microscope. Results We found that ADM-induced enhancement of BRR response was blunted by microinjection of NPLA or Rp-8-Br-cGMP, a selective inhibitor of nNOS or protein kinase G (PKG respectively, into NTS. Western blot analysis further revealed that ADM induced an increase in the protein level of PKG-I which could be attenuated by co-microinjection with the ADM receptor antagonist ADM22-52 or NPLA. Moreover, we observed an increase in phosphorylation at Ser1416 of nNOS at 10, 30, and 60 min after intra-NTS administration of ADM. As such, nNOS/PKG signaling may also account for the enhancing effect of ADM on BRR response. Interestingly, biochemical evidence further showed that ADM-induced increase of nNOS phosphorylation was prevented by co-microinjection with Rp-8-Br-cAMP, a PKA inhibitor. The possibility of PKA-dependent nNOS activation was substantiated by immunohistochemical demonstration of co-localization of PKA and nNOS in putative NTS neurons. Conclusions The novel finding of this study is that the signal transduction cascade that

  5. Strigolactone Levels in Dicot Roots Are Determined by an Ancestral Symbiosis-Regulated Clade of the PHYTOENE SYNTHASE Gene Family

    Directory of Open Access Journals (Sweden)

    Ron Stauder

    2018-03-01

    Full Text Available Strigolactones (SLs are apocarotenoid phytohormones synthesized from carotenoid precursors. They are produced most abundantly in roots for exudation into the rhizosphere to cope with mineral nutrient starvation through support of root symbionts. Abscisic acid (ABA is another apocarotenoid phytohormone synthesized in roots, which is involved in responses to abiotic stress. Typically low carotenoid levels in roots raise the issue of precursor supply for the biosynthesis of these two apocarotenoids in this organ. Increased ABA levels upon abiotic stress in Poaceae roots are known to be supported by a particular isoform of phytoene synthase (PSY, catalyzing the rate-limiting step in carotenogenesis. Here we report on novel PSY3 isogenes from Medicago truncatula (MtPSY3 and Solanum lycopersicum (SlPSY3 strongly expressed exclusively upon root interaction with symbiotic arbuscular mycorrhizal (AM fungi and moderately in response to phosphate starvation. They belong to a widespread clade of conserved PSYs restricted to dicots (dPSY3 distinct from the Poaceae-PSY3s involved in ABA formation. An ancient origin of dPSY3s and a potential co-evolution with the AM symbiosis is discussed in the context of PSY evolution. Knockdown of MtPSY3 in hairy roots of M. truncatula strongly reduced SL and AM-induced C13 α-ionol/C14 mycorradicin apocarotenoids. Inhibition of the reaction subsequent to phytoene synthesis revealed strongly elevated levels of phytoene indicating induced flux through the carotenoid pathway in roots upon mycorrhization. dPSY3 isogenes are coregulated with upstream isogenes and downstream carotenoid cleavage steps toward SLs (D27, CCD7, CCD8 suggesting a combined carotenoid/apocarotenoid pathway, which provides “just in time”-delivery of precursors for apocarotenoid formation.

  6. Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response

    KAUST Repository

    Almadanim, M. Cecília

    2017-01-19

    Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analyzing OsCPK17 knockout, silencing, and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress-inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose phosphate synthase OsSPS4, and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium-dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.

  7. Molecular cloning and expression levels of the monoterpene synthase gene (ZMM1) in Cassumunar ginger (Zingiber montanum (Koenig) Link ex Dietr.)

    OpenAIRE

    Bua-In Saowaluck; Paisooksantivatana Yingyong; Weimer Bart C.; Chowpongpang Srimek

    2014-01-01

    Cassumunar ginger (Zingiber montanum (Koenig) Link ex Dietr.) is a native Thai herb with a high content and large variety of terpenoids in its essential oil. Improving the essential oil content and quality of cassumunar ginger is difficult for a breeder due to its clonally propagated nature. In this research, we describe the isolation and expression level of the monoterpene synthase gene that controls the key step of essential oil synthesis in this plant an...

  8. Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis1[OPEN

    Science.gov (United States)

    Zhang, Chunyu; Zhang, Wei; Ren, Guodong; Li, Delin; Cahoon, Rebecca E.; Chen, Ming; Zhou, Yongming; Yu, Bin

    2015-01-01

    Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis. PMID:26048882

  9. Contribution of granule bound starch synthase in kernel modification ...

    African Journals Online (AJOL)

    The role of gbssI and gbssII genes, encoding granule bound starch synthase enzyme I and II, respectively, in quality protein maize (QPM) were studied at different days after pollination (DAP). Total RNA was used for first strand cDNA synthesis using the ImpromIISriptTM reverse transcriptase. No detectable levels of gbssI ...

  10. A CACGTG motif of the Antirrhinum majus chalcone synthase promoter is recognized by an evolutionarily conserved nuclear protein

    International Nuclear Information System (INIS)

    Staiger, D.; Kaulen, H.; Schell, J.

    1989-01-01

    In the chalcone synthase gene of Antirrhinum majus (snapdragon), 150 base pairs of the 5' flanking region contain cis-acting signals for UV light-induced expression. A nuclear factor, designated CG-1, specifically recognizes a hexameric motif with internal dyad symmetry, CACGTG, located within this light-responsive sequence. Binding of CG-1 is influenced by C-methylation of the CpG dinucleotide in the recognition sequence. CG-1 is a factor found in a variety of dicotyledonous plant species including Nicotiana tabacum, A. majus, Petunia hybrida, Arabidopsis thaliana, and Glycine max. CACGTG motifs contained within trans-acting factor recognition sites in various other plant promoters can interact with CG-1. In addition, the binding site of the human adenovirus major late transcription factor USF can compete for CG-1 binding to the chalcone synthase promoter. This suggests an evolutionary conservation of trans-acting factor recognition sites involved in divergent mechanisms of gene control. (author)

  11. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  12. Probing the Selectivity and Protein•Protein Interactions of a Non-Reducing Fungal Polyketide Synthase Using Mechanism-Based Crosslinkers

    Science.gov (United States)

    Bruegger, Joel; Haushalter, Bob; Vagstad, Anna; Shakya, Gaurav; Mih, Nathan; Townsend, Craig A.; Burkart, Michael D.; Tsai, Shiou-Chuan

    2013-01-01

    SUMMARY Protein•protein interactions, which often involve interactions between an acyl carrier protein (ACP) and its partner enzymes, are important for coordinating polyketide biosynthesis. However, the nature of such interactions is not well understood, especially in the fungal non-reducing polyketide synthases (NR-PKSs) that biosynthesize toxic and pharmaceutically important polyketides. Here, we employ a mechanism-based crosslinker to successfully probe ACP and ketosynthase (KS) domain interactions in NR-PKSs. We found that crosslinking efficiency is closely correlated with the strength of ACP•KS interactions, and that KS demonstrates strong starter unit selectivity. We further identified positively charged surface residues by KS mutagenesis, which mediate key interactions with the negatively-charged ACP surface. Such complementary/matching contact pairs can serve as “adapter surfaces” for future efforts to generate new polyketides using NR-PKSs. PMID:23993461

  13. Lower Squalene Epoxidase and Higher Scavenger Receptor Class B Type 1 Protein Levels Are Involved in Reduced Serum Cholesterol Levels in Stroke-Prone Spontaneously Hypertensive Rats.

    Science.gov (United States)

    Michihara, Akihiro; Mido, Mayuko; Matsuoka, Hiroshi; Mizutani, Yurika

    2015-01-01

    A lower serum cholesterol level was recently shown to be one of the causes of stroke in an epidemiological study. Spontaneously hypertensive rats stroke-prone (SHRSP) have lower serum cholesterol levels than normotensive Wistar-Kyoto rats (WKY). To elucidate the mechanisms responsible for the lower serum cholesterol levels in SHRSP, we determined whether the amounts of cholesterol biosynthetic enzymes or the receptor and transporter involved in cholesterol uptake and efflux in the liver were altered in SHRSP. When the mRNA levels of seven cholesterol biosynthetic enzymes were measured using real-time polymerase chain reaction (PCR), farnesyl pyrophosphate synthase and squalene epoxidase (SQE) levels in the liver of SHRSP were significantly lower than those in WKY. SQE protein levels were significantly reduced in tissues other than the brain of SHRSP. No significant differences were observed in low-density lipoprotein (LDL) receptor (uptake of serum LDL-cholesterol) or ATP-binding cassette transporter A1 (efflux of cholesterol from the liver/formation of high-density lipoprotein (HDL)) protein levels in the liver and testis between SHRSP and WKY, whereas scavenger receptor class B type 1 (SRB1: uptake of serum HDL-cholesterol) protein levels were higher in the livers of SHRSP. These results indicated that the lower protein levels of SQE and higher protein levels of SRB1 in the liver were involved in the reduced serum cholesterol levels in SHRSP.

  14. Sucrose-phosphate synthase in tree species: light/dark regulation involves a component of protein turnover in Prosopis juliflora (SW DC).

    Science.gov (United States)

    Sinha, A K; Shirke, P A; Pathre, U; Sane, P V

    1997-10-01

    Light dependent modulation of sucrose-phosphate synthase activity (SPS; EC 2.4.1.14) was studied in a tree species, namely Prosopis juliflora. In this paper we demonstrate that cycloheximide, an inhibitor of cytoplasmic protein synthesis, when fed to detached leaves of P. juliflora through transpiration stream in the dark or in light completely prevents in vivo light activation of Vlim and Vmax activities of SPS. In case of spinach, however, cycloheximide feeding affects only Vlim activity while Vmax activity remained unchanged. In contrast, chloramphenicol, an inhibitor of protein synthesis in chloroplast has no effect on the light activation of SPS in Prosopis. The treatment with cycloheximide showed slight reduction in the rate of O2 evolution indicating that cycloheximide had very little effect on overall photosynthesis. These results indicate that short term protein turnover of the SPS protein and some other essential component(s) (e.g., a putative protein that modifies SPS activity) is one of the primary steps in a complex and unique regulatory cascade effecting the reversible light activation of SPS.

  15. Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome.

    Science.gov (United States)

    Müller, Christina A; Oberauner-Wappis, Lisa; Peyman, Armin; Amos, Gregory C A; Wellington, Elizabeth M H; Berg, Gabriele

    2015-08-01

    Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Diffusible signal factor (DSF) synthase RpfF of Xylella fastidiosa is a multifunction protein also required for response to DSF.

    Science.gov (United States)

    Ionescu, Michael; Baccari, Clelia; Da Silva, Aline Maria; Garcia, Angelica; Yokota, Kenji; Lindow, Steven E

    2013-12-01

    Xylella fastidiosa, like related Xanthomonas species, employs an Rpf cell-cell communication system consisting of a diffusible signal factor (DSF) synthase, RpfF, and a DSF sensor, RpfC, to coordinate expression of virulence genes. While phenotypes of a ΔrpfF strain in Xanthomonas campestris could be complemented by its own DSF, the DSF produced by X. fastidiosa (XfDSF) did not restore expression of the XfDSF-dependent genes hxfA and hxfB to a ΔrpfF strain of X. fastidiosa, suggesting that RpfF is involved in XfDSF sensing or XfDSF-dependent signaling. To test this conjecture, rpfC and rpfF of X. campestris were replaced by those of X. fastidiosa, and the contribution of each gene to the induction of a X. campestris DSF-dependent gene was assessed. As in X. fastidiosa, XfDSF-dependent signaling required both X. fastidiosa proteins RpfF and RpfC. RpfF repressed RpfC signaling activity, which in turn was derepressed by XfDSF. A mutated X. fastidiosa RpfF protein with two substitutions of glutamate to alanine in its active site was incapable of XfDSF production yet enabled a response to XfDSF, indicating that XfDSF production and the response to XfDSF are two separate functions in which RpfF is involved. This mutant was also hypervirulent to grape, demonstrating the antivirulence effects of XfDSF itself in X. fastidiosa. The Rpf system of X. fastidiosa is thus a novel example of a quorum-sensing signal synthase that is also involved in the response to the signal molecule that it synthesizes.

  17. Protein C activity and antigen levels in childhood

    NARCIS (Netherlands)

    van Teunenbroek, A.; Peters, M.; Sturk, A.; Borm, J. J.; Breederveld, C.

    1990-01-01

    Hereditary protein C deficiency is an important risk factor for thrombosis. To enable its diagnosis shortly after birth, we determined reference values of protein C antigen and activity levels for the first 3 months of life. To establish an age-related range of protein C levels we also determined

  18. PROTEIN TARGETING TO STARCH is required for localising GRANULE-BOUND STARCH SYNTHASE to starch granules and for normal amylose synthesis in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    David Seung

    2015-02-01

    Full Text Available The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin or linear (amylose. The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM. We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is

  19. Analogies and surprising differences between recombinant nitric oxide synthase-like proteins from Staphylococcus aureus and Bacillus anthracis in their interactions with l-arginine analogs and iron ligands.

    Science.gov (United States)

    Salard, Isabelle; Mercey, Emilie; Rekka, Eleni; Boucher, Jean-Luc; Nioche, Pierre; Mikula, Ivan; Martasek, Pavel; Raman, C S; Mansuy, Daniel

    2006-12-01

    Genome sequencing has recently shown the presence of genes coding for NO-synthase (NOS)-like proteins in bacteria. The roles of these proteins remain unclear. The interactions of a series of l-arginine (l-arg) analogs and iron ligands with two recombinant NOS-like proteins from Staphylococcus aureus (saNOS) and Bacillus anthracis (baNOS) have been studied by UV-visible spectroscopy. SaNOS and baNOS in their ferric native state, as well as their complexes with l-arg analogs and with various ligands, exhibit spectral characteristics highly similar to the corresponding complexes of heme-thiolate proteins such as cytochromes P450 and NOSs. However, saNOS greatly differs from baNOS at the level of three main properties: (i) native saNOS mainly exists under an hexacoordinated low-spin ferric state whereas native baNOS is mainly high-spin, (ii) the addition of tetrahydrobiopterin (H4B) or H4B analogs leads to an increase of the affinity of l-arg for saNOS but not for baNOS, and (iii) saNOS Fe(II), contrary to baNOS, binds relatively bulky ligands such as nitrosoalkanes and tert-butylisocyanide. Thus, saNOS exhibits properties very similar to those of the oxygenase domain of inducible NOS (iNOS(oxy)) not containing H4B, as expected for a NOSoxy-like protein that does not contain H4B. By contrast, the properties of baNOS which look like those of H4B-containing iNOS(oxy) are unexpected for a NOS-like protein not containing H4B. The origin of these surprising properties of baNOS remains to be determined.

  20. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    International Nuclear Information System (INIS)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S.

    1991-01-01

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[ 35 S]methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate

  1. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  2. The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice

    Science.gov (United States)

    Atochina-Vasserman, Elena N.; Massa, Christopher B.; Birkelbach, Bastian; Guo, Chang-Jiang; Scott, Pamela; Haenni, Beat; Beers, Michael F.; Ochs, Matthias; Gow, Andrew J.

    2015-01-01

    Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd−/−) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd−/− mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd−/− mice. These changes were reduced in DiNOS, and compared with Sftpd−/− mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd−/−. Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces. PMID:26320150

  3. Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.

    Science.gov (United States)

    Wang, Shuxin; Li, Zheyi; Shen, Hongyan; Zhang, Zhong; Yin, Yuxin; Wang, Qingsong; Zhao, Xuyang; Ji, Jianguo

    2016-08-01

    Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3β (Gsk3β). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101. © 2016 AlphaMed Press.

  4. Effects of increasing dietary protein levels on growth, feed utilization ...

    African Journals Online (AJOL)

    Yomi

    2012-01-05

    Jan 5, 2012 ... The effect of different dietary protein levels on growth performance and on feed utilization of catfish. (Heterobranchus ... (Legendre, 1991) because of its taste, fast growth rate ..... diet containing 40% protein had high growth with low food intake and feed ... protein rate (45%) combined with a bad utilization of.

  5. Changes in Serum Proteins and Creatinine levels in HIV Infected ...

    African Journals Online (AJOL)

    This study examined the level of total serum proteins and globulins in HIV infected Nigerians. 64 patients with HIV infection and 10 apparently healthy subjects were recruited from 3 hospitals in Lagos Metropolis. They were examined for the presence of TB and malaria. Serum total protein, albumin and creatinine levels ...

  6. β-chain of ATP synthase as a lipophorin binding protein and its role in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae).

    Science.gov (United States)

    Fruttero, Leonardo L; Demartini, Diogo R; Rubiolo, Edilberto R; Carlini, Célia R; Canavoso, Lilián E

    2014-09-01

    Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The β subunit of the ATP synthase complex (β-ATPase) was identified as a lipophorin binding protein. β-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that β-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and β-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of β-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the β-ATPase antibody significantly reduced the transfer of [(3)H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-(3)H]-oleic acid, supporting the involvement of lipophorin-β-ATPase association in the transfer of lipids. In addition, the β-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Nitric oxide synthesis-promoting effects of valsartan in human umbilical vein endothelial cells via the Akt/adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

    Directory of Open Access Journals (Sweden)

    Yingshuai Zhao

    2017-05-01

    Full Text Available Valsartan (VAL, an antagonist of angiotensin II receptor type 1, has antihypertensive and multiple cardiovascular protective effects. The pleiotropic functions of VAL are related to the increased synthesis and biological activity of intravascular nitric oxide (NO. In this study, the role and mechanisms of VAL in the synthesis of NO were examined in human umbilical vein endothelial cells (HUVECs. Ten µmol/L of VAL was used to treat EA.hy926 cells for 30 minutes, 1, 3, 6, 12, and 24 hours, and three concentrations of VAL (i.e., 10, 1, and 0.1 µmol/L were used to treat EA.hy926 cells for 24 hours. The cells were divided into five groups: control, VAL, VAL + Compound C (adenosine monophosphate-activated protein kinase [AMPK] inhibitor, 1 µmol/L, VAL + LY294002 (Akt [protein kinase B] inhibitor, 10 µmol/L, and VAL + L-nitro-arginine methyl ester (L-NAME, endothelial NO synthase [eNOS] inhibitor, 500 µmol/L groups. The NO content in the VAL-treated HUVEC line (EA.hy926 was detected using the nitrate reductase method, and western blot was used to detect the phosphorylation of Akt, AMPK, and eNOS, as well as the changes in total protein levels. VAL increased NO synthesis in EA.hy926 cells in time- and dose-dependent manners (p < 0.05 and the intracellular phosphorylation levels of Akt, AMPK, and eNOS at the corresponding time points. LY294002, Compound C, and L-NAME could inhibit the VAL-promoted NO synthesis. VAL activated Akt, AMPK, and eNOS, thus promoting NO synthesis and playing a protective role in endothelial cells. These results partially explained the mechanisms underlying the cardiovascular protective effects of VAL.

  8. Identification of an abundant 56 kDa protein implicated in food allergy as granule-bound starch synthase

    Science.gov (United States)

    Rice, the staple food of South and East Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16 kDa, 26 kDa, 33 kDa and 56 kDa have been identified as allergens. Re...

  9. The Structure of Rauvolfia serpentina Strictosidine Synthase Is a Novel Six-Bladed β-Propeller Fold in Plant Proteins[W

    Science.gov (United States)

    Ma, Xueyan; Panjikar, Santosh; Koepke, Juergen; Loris, Elke; Stöckigt, Joachim

    2006-01-01

    The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of ∼2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler–type reaction and represents a novel six-bladed β-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed β-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family. PMID:16531499

  10. Attenuation of ischemia-reperfusion injury by sevoflurane postconditioning involves protein kinase B and glycogen synthase kinase 3 beta activation in isolated rat hearts.

    Science.gov (United States)

    Fang, Neng-Xin; Yao, Yun-Tai; Shi, Chun-Xia; Li, Li-Huan

    2010-12-01

    Volatile anesthetic ischemic postconditioning reduces infarct size following ischemia/reperfusion. Whether phosphorylation of protein kinase B (PKB/Akt) and glycogen synthase kinase 3 beta (GSK3β) is causal for cardioprotection by postconditioning is controversial. We therefore investigated the impact of PKB/Akt and GSK3β in isolated perfused rat hearts subjected to 40 min of ischemia followed by 1 h of reperfusion. 2.0% sevoflurane (1.0 minimum alveolar concentration) was administered at the onset of reperfusion in 15 min as postconditioning. Western blot analysis was used to determine phosphorylation of PKB/Akt and its downstream target GSK3β after 1 h of reperfusion. Mitochondrial and cytosolic content of cytochrome C checked by western blot served as a marker for mitochondrial permeability transition pore opening. Sevoflurane postconditioning significantly improved functional cardiac recovery and decreased infarct size in isolated rat hearts. Compared with unprotected hearts, sevoflurane postconditioning-induced phosphorylation of PKB/Akt and GSK3β were significantly increased. Increase of cytochrome C in mitochondria and decrease of it in cytosol is significant when compared with unprotected ones which have reversal effects on cytochrome C. The current study presents evidence that sevoflurane-induced cardioprotection at the onset of reperfusion are partly through activation of PKB/Akt and GSK3β.

  11. The role of surface electrostatics on the stability, function and regulation of human cystathionine β-synthase, a complex multidomain and oligomeric protein.

    Science.gov (United States)

    Pey, Angel L; Majtan, Tomas; Kraus, Jan P

    2014-09-01

    Human cystathionine β-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability are independently regulated by the binding of S-adenosyl-methionine (SAM) to two different types of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Crocin Suppresses LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by Upregulation of Heme Oxygenase-1 via Calcium/Calmodulin-Dependent Protein Kinase 4

    Directory of Open Access Journals (Sweden)

    Ji-Hee Kim

    2014-01-01

    Full Text Available Crocin is a water-soluble carotenoid pigment that is primarily used in various cuisines as a seasoning and coloring agent, as well as in traditional medicines for the treatment of edema, fever, and hepatic disorder. In this study, we demonstrated that crocin markedly induces the expression of heme oxygenase-1 (HO-1 which leads to an anti-inflammatory response. Crocin inhibited inducible nitric oxide synthase (iNOS expression and nitric oxide production via downregulation of nuclear factor kappa B activity in lipopolysaccharide- (LPS- stimulated RAW 264.7 macrophages. These effects were abrogated by blocking of HO-1 expression or activity. Crocin also induced Ca2+ mobilization from intracellular pools and phosphorylation of Ca2+/calmodulin-dependent protein kinase 4 (CAMK4. CAMK4 knockdown and kinase-dead mutant inhibited crocin-mediated HO-1 expression, Nrf2 activation, and phosphorylation of Akt, indicating that HO-1 expression is mediated by CAMK4 and that Akt is a downstream mediator of CAMK4 in crocin signaling. Moreover, crocin-mediated suppression of iNOS expression was blocked by CAMK4 inhibition. Overall, these results suggest that crocin suppresses LPS-stimulated expression of iNOS by inducing HO-1 expression via Ca2+/calmodulin-CAMK4-PI3K/Akt-Nrf2 signaling cascades. Our findings provide a novel molecular mechanism for the inhibitory effects of crocin against endotoxin-mediated inflammation.

  13. Glycogen synthase kinase-3beta (GSK3beta) negatively regulates PTTG1/human securin protein stability, and GSK3beta inactivation correlates with securin accumulation in breast tumors.

    Science.gov (United States)

    Mora-Santos, Mar; Limón-Mortés, M Cristina; Giráldez, Servando; Herrero-Ruiz, Joaquín; Sáez, Carmen; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

    2011-08-26

    PTTG1, also known as securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for securin in tumor progression. Stability of securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting securin for proteolysis have not been identified, and mechanistic insight into the cause of securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates securin to promote its proteolysis via SCF(βTrCP) E3 ubiquitin ligase. Importantly, a strong correlation between securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for securin accumulation in breast cancers.

  14. The clinical expression of hereditary protein C and protein S deficiency: : a relation to clinical thrombotic risk-factors and to levels of protein C and protein S

    NARCIS (Netherlands)

    Henkens, C. M. A.; van der Meer, J.; Hillege, J. L.; Bom, V. J. J.; Halie, M. R.; van der Schaaf, W.

    We investigated 103 first-degree relatives of 13 unrelated protein C or protein S deficient patients to assess the role of additional thrombotic risk factors and of protein C and protein S levels in the clinical expression of hereditary protein C and protein S deficiency. Fifty-seven relatives were

  15. Dephosphorylation of the Core Clock Protein KaiC in the Cyanobacterial KaiABC Circadian Oscillator Proceeds via an ATP Synthase Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Egli, Martin; Mori, Tetsuya; Pattanayek, Rekha; Xu, Yao; Qin, Ximing; Johnson, Carl H. (Vanderbilt)

    2014-10-02

    The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P{sub i} (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the

  16. Impact of Catheter Arteriography on the Serum Level of Asymmetric Dimethylarginine, an Endogenous Inhibitor of Nitric Oxide Synthase

    International Nuclear Information System (INIS)

    Bozlar, Ugur; Ugurel, Mehmet Sahin; Ozcan, Omer; Cakir, Erdinc; Ustunsoz, Bahri; Ucoz, Taner; Bilgi, Cumhur; Somuncu, Ibrahim

    2008-01-01

    The purpose of this study was to investigate the instantaneous impact of catheter arteriography on blood asymmetric dimethylarginine (ADMA) levels in accordance with patient- and procedure-related variables. Sixty-eight patients (16 women, 52 men; mean age, 45.6 ± 20.1 years; range, 20-79 years) referred for cerebral or peripheral catheter arteriography were recruited for the study. Pre- and postarteriography arterial blood ADMA levels were determined by high-performance liquid chromatographic technique. Type of nonionic iodinated contrast media used, duration of procedure, patient gender, and patient age were noted and evaluated as possible factors that could influence serum ADMA levels in arteriography procedures. Prearteriography ADMA levels decreased significantly after arteriography in general (pre, 1.16 ± 0.96 μmol/L; post, 1.08 ± 0.80 μmol/L; p = 0.002). Males tended to have lower postarteriography serum ADMA levels (p = 0.005). Serum ADMA levels tended to get lower after peripheral arteriography procedures (p = 0.005) and when iohexol, 350 mg I/ml, was used as the contrast agent (p = 0.017). In conclusion, ADMA level does not seem to be subject to acute elevation after catheter arteriography; on the contrary, its level may decrease in general. Moreover, a reduction in serum ADMA level may be expected, especially in male patients, in patients who undergo a peripheral arteriography procedure, or when iohexol, 350 mg I/ml, is used as the contrast agent

  17. Transcriptional regulation of fksA, a β-1,3-glucan synthase gene, by the APSES protein StuA during Aspergillus nidulans development.

    Science.gov (United States)

    Park, Bum-Chan; Park, Yun-Hee; Yi, Soohyun; Choi, Yu Kyung; Kang, Eun-Hye; Park, Hee-Moon

    2014-11-01

    The temporal and spatial regulation of β-1,3-glucan synthesis plays an important role in morphogenesis during fungal growth and development. Northern blot analysis showed that the transcription of fksA, the gene encoding β-1,3-glucan synthase in Aspergillus nidulans, was cell-cycle-dependent and increased steadily over the duration of the vegetative period, but its overall expression during the asexual and sexual stages was fairly constant up until the time of transcription cessation. In an A. nidulans strain mutated in the eukaryotic bHLH-like APSES transcription factor stuA1, the transcriptional level of fksA, and consequently the content of alkali-insoluble cell wall β-glucan, significantly increased at the conidial chain formation and maturation stage. Electrophoretic mobility shift assays revealed that StuA was bound to StREs (StuA Response Elements) on the fksA promoter region. Promoter analysis with sGFP-fusion constructs also indicated the negative regulation of fksA expression by StuA, especially during asexual development. Taken together, these data suggest that StuA plays an important role in cell wall biogenesis during the development of A. nidulans, by controlling the transcription level of fksA.

  18. Microsatellite instability and the association with plasma homocysteine and thymidylate synthase in colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Lars Henrik; Lindebjerg, Jan; Crüger, Dorthe G.

    2008-01-01

    , carcinoembryonic antigen, vitamin B12, and folate. Microsatellite instability of tumors was associated with higher levels of plasma homocysteine (p = 0.008) and higher protein expression of thymidylate synthase (p ... factors. CEA was not associated with neither homocysteine nor microsatellite instability. The data suggests that there is a more pronounced methyl unit deficiency in microsatellite instable tumors....

  19. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    Science.gov (United States)

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. A 31 bp VNTR in the cystathionine beta-synthase (CBS) gene is associated with reduced CBS activity and elevated post-load homocysteine levels.

    NARCIS (Netherlands)

    Lievers, K.J.; Kluijtmans, L.A.J.; Heil, S.G.; Boers, G.H.J.; Verhoef, P.; Oppenraaij-Emmerzaal, D. van; Heijer, M. den; Trijbels, J.M.F.; Blom, H.J.

    2001-01-01

    Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinaemia, an independent and graded risk factor for cardiovascular disease (CVD). Although heterozygosity for cystathionine beta-synthase (CBS) deficiency has been excluded as a major

  1. A 31 bp VNTR in the cystathionine beta-synthase (CBS) gene is associated with reduced CBS activity and elevated post-load homocysteine levels

    NARCIS (Netherlands)

    Lievers, K.J.; Kluijtmans, L.A.; Heil, S.G.; Boers, G.H.J.; Verhoef, P.; Oppenraay-Emmerzaal, van D.; Heijer, den M.; Trijbels, F.J.M.; Blom, H.J.

    2001-01-01

    Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinaemia, an independent and graded risk factor for cardiovascular disease (CVD). Although heterozygosity for cystathionine -synthase (CBS) deficiency has been excluded as a major

  2. Dietary protein level and performance of growing Baladi kids.

    Science.gov (United States)

    Abdelrahman, M M; Aljumaah, R S

    2014-01-01

    A study was conducted to evaluate the effect of feeding different levels of protein to black Baladi breed kids. Weanling Baladi kids (n=18; 75 to 90 days old) were selected and individually housed at our experimental farm. Kids were divided randomly to one of the three treatments for 12 weeks. The three dietary treatments were: T1: control ration, formulated according to NRC to cover the protein (level 1) and other nutrients requirements. T2: ration formulated to cover only 75% of protein (level 2) recommended by NRC. T3: control diet + 2.4 g undegradable methionine (Smartamine®)/day/kid (level 3). Feed intake, initial and monthly body weights were recorded. Blood samples were collected monthly and analyzed for metabolites and Co, Zn and Cu levels. Decreasing the dietary level of protein (T2) negatively affected (Pkids below the NRC requirements of protein negatively affect the growth performance and feed efficiency. The recommended protein level by NRC for growing kids cover the requirements of growing black Baladi kids for maximum growth and productivity.

  3. Effect Of Dietary Protein Levels On The Performance And Carcass ...

    African Journals Online (AJOL)

    Effect Of Dietary Protein Levels On The Performance And Carcass ... Nigerian Journal of Animal Production ... Response criteria such as weight gain and feed conversion ratio, among others, and carcass characteristics were measured.

  4. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-α-dependent pathway in human dermal fibroblasts

    International Nuclear Information System (INIS)

    Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi

    2011-01-01

    Highlights: ► Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. ► Adiponectin also increases the phosphorylation of AMPK. ► A pharmacological activator of AMPK increases mRNA levels of PPARα and HAS2. ► Adiponectin-induced HAS2 mRNA expression is blocked by a PPARα antagonist. ► Adiponectin promotes hyaluronan synthesis via an AMPK/PPARα-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1β-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-α (PPARα), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPARα antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPARα-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  5. Development of a biomarker for Geobacter activity and strain composition: Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI)

    Energy Technology Data Exchange (ETDEWEB)

    Wilkins, M.J.; Callister, S.J.; Miletto, M.; Williams, K.H.; Nicora, C.D.; Lovley, D.R.; Long, P.E.; Lipton, M.S.

    2010-02-15

    Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

  6. Effects of indomethacin, NS-398 (a selective prostaglandin H synthase-2 inhibitor) and protein synthesis inhibitors on prostaglandin production by the guinea-pig placenta.

    Science.gov (United States)

    Aitken, H; Poyser, N L

    2001-01-01

    The outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)were similar from the day 22 guinea-pig placenta and sub-placenta in culture, except for PGE2 output from the sub-placenta which was lower. Between days 22 and 29 of pregnancy, the outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)during the initial 2 h culture period increased 6.9-, 1.1- and 3.2-fold, respectively, from the placenta, and 2.1-, 1.4- and 2.2-fold, respectively, from the sub-placenta. Therefore, there was a relatively specific increase in PGF(2 alpha)production by the guinea-pig placenta between days 22 and 29 of pregnancy. The output of PGFM from the cultured placenta also increased between days 22 and 29, indicating that the increase in PGF(2 alpha)output was due to increased synthesis rather than to decreased metabolism. By comparing the amounts of prostaglandins produced by tissue homogenates during a 1 h incubation period, it appears that there is approximately a 2-fold increase in the amount of prostaglandin H synthase (PGHS) present in the guinea-pig placenta between days 22 and 29. NS-398 (a specific inhibitor of PGHS-2) and indomethacin (an inhibitor of both PGHS-1 and PGHS-2) both inhibited prostaglandin production by homogenates of day 22 and day 29 placenta. Indomethacin was more effective than NS-398, except for their actions on PGF(2 alpha)production by the day 29 placenta where indomethacin and NS-398 were equiactive. Indomethacin and NS-398 were both very effective at inhibiting the outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)from the day 22 and day 29 placenta and sub-placenta in culture, indicating that prostaglandin production by the guinea-pig placenta and sub-placenta in culture is largely dependent upon the activity of PGHS-2. The high production of PGF(2 alpha)by the day 29 placenta is not dependent on the continual synthesis of fresh protein(s), as inhibitors of protein synthesis did not reduce PGF(2 alpha)output from the day 29 guinea-pig placenta in culture

  7. Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response

    KAUST Repository

    Almadanim, M. Cecí lia; Alexandre, Bruno M.; Rosa, Margarida T.G.; Sapeta, Helena; Leitã o, Antó nio E.; Ramalho, José C.; Lam, TuKiet T.; Negrã o, Só nia; Abreu, Isabel A.; Oliveira, M. Margarida

    2017-01-01

    Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here

  8. Protein Expression Analyses at the Single Cell Level

    Directory of Open Access Journals (Sweden)

    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  9. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    International Nuclear Information System (INIS)

    Hwang, Yong Pil; Kim, Hyung Gyun; Hien, Tran Thi; Jeong, Myung Ho; Jeong, Tae Cheon; Jeong, Hye Gwang

    2011-01-01

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-α-stimulated monocytes to endothelial cells and suppressed the TNF-α induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-α-induced nuclear factor-κB activation, which was attenuated by pretreatment with N G -nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: ► Puerarin induced the phosphorylation of eNOS and the production of NO. ► Puerarin activated eNOS through ER-dependent PI3-kinase and Ca 2+ -dependent AMPK. ► Puerarin-induced NO was involved in the inhibition of NF-kB activation. ► Puerarin may help for prevention of vascular dysfunction and diabetes.

  10. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Yong Pil; Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Hien, Tran Thi [College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Jeong, Myung Ho [Heart Research Center, Chonnam National University Hospital, Gwangju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyungsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  11. Low sulfide levels and a high degree of cystathionine β-synthase (CBS) activation by S-adenosylmethionine (SAM) in the long-lived naked mole-rat.

    Science.gov (United States)

    Dziegelewska, Maja; Holtze, Susanne; Vole, Christiane; Wachter, Ulrich; Menzel, Uwe; Morhart, Michaela; Groth, Marco; Szafranski, Karol; Sahm, Arne; Sponholz, Christoph; Dammann, Philip; Huse, Klaus; Hildebrandt, Thomas; Platzer, Matthias

    2016-08-01

    Hydrogen sulfide (H2S) is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine β-synthase (CBS), an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Low sulfide levels and a high degree of cystathionine β-synthase (CBS activation by S-adenosylmethionine (SAM in the long-lived naked mole-rat

    Directory of Open Access Journals (Sweden)

    Maja Dziegelewska

    2016-08-01

    Full Text Available Hydrogen sulfide (H2S is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine β-synthase (CBS, an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration.

  13. Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.

    Science.gov (United States)

    Beekwilder, Jules; van Houwelingen, Adèle; Cankar, Katarina; van Dijk, Aalt D J; de Jong, René M; Stoopen, Geert; Bouwmeester, Harro; Achkar, Jihane; Sonke, Theo; Bosch, Dirk

    2014-02-01

    Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  14. Far-infrared radiation acutely increases nitric oxide production by increasing Ca(2+) mobilization and Ca(2+)/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179.

    Science.gov (United States)

    Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

    2013-07-12

    Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser(1179)) in a time-dependent manner (up to 40min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca(2+) levels. Treatment with KN-93, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. This study suggests that FIR radiation increases NO production via increasing CaMKII-mediated eNOS-Ser(1179) phosphorylation but TRPV channels may not be involved in this pathway. Our results may provide the molecular mechanism by which FIR radiation improves endothelial function. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Decreased hippocampal homoarginine and increased nitric oxide and nitric oxide synthase levels in rats parallel training in a radial arm maze.

    Science.gov (United States)

    Sase, Ajinkya; Nawaratna, Gayan; Hu, Shengdi; Wu, Guoyao; Lubec, Gert

    2016-09-01

    L-homoarginine (hArg) is derived from enzymatic guanidination of lysine. It was demonstrated that hArg is a substrate for nitric oxide (NO) synthesis, blocks lysine transport and inhibits the uptake of arginine into synaptosomes and modulates GABA responses ex vivo. As there is limited information on its physiological roles in the brain, the aim of the study was to show whether hippocampal or frontal lobe (FL) hArg is paralleling training in the radial arm maze (RAM) or NO formation. Hippocampi and FL of male Sprague-Dawley rats were taken from trained or yoked in a RAM. Then hArg and metabolites, NO and NO synthase (NOS) were determined by standard methods. The animals learned the task in the RAM showing significant reduction of working memory errors. hArg showed decreased levels in both brain regions of trained animals as compared to yoked animals. Nitrate plus nitrite (NOx) concentrations and NOS activity were significantly increased in hippocampi, F(1,36) = 170.5; P ≤ 0.0001 and FL, F(1,36) = 74.67; P ≤ 0.0001 of trained animals as compared to yoked animals. Levels of hArg were negatively correlated with NOx in hippocampus (r = -0.6355; P = 0.0483) but not in FL and with lysine in the FL (r = -0.6650; P = 0.0358). NOx levels were positively correlated with NOS in both the hippocampus (r = 0.7474; P = 0.0129) and FL (r = 0.9563; P ≤  0.0001). These novel findings indicate that hArg is linked to NO formation in hippocampus but not in FL and is paralleling spatial memory in the RAM.

  16. HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels.

    Science.gov (United States)

    Bruno, Anna Paola; De Simone, Francesca Isabella; Iorio, Vittoria; De Marco, Margot; Khalili, Kamel; Sariyer, Ilker Kudret; Capunzo, Mario; Nori, Stefania Lucia; Rosati, Alessandra

    2014-01-01

    BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.

  17. Changes in angiotensin AT1 receptor mRNA levels in the rat brain after immobilization stress and inhibition of central nitric oxide synthase.

    Science.gov (United States)

    Kiss, A; Jurkovicova, D; Jezova, D; Krizanova, O

    2001-06-01

    To study functional interactions between angiotensin II AT1 receptors and nitric oxide (NO) activity in different brain areas in rats exposed to immobilization stress. Central inhibition of nitric oxide synthase (NOS) was provided by intracerebroventricular (i.c.v.) administration of (N-omega-nitro-L-arginine-methylester) L-NAME and analysis of AT1 receptor mRNA was performed using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. The immobilization in prone position lasted 2 hrs and the rats were sacrificed 24 hr later. The hypothalamus, hippocampus, thalamus, and cortex were isolated from fresh brains. In the cortex, gene expression of AT1 receptors was unaffected either by L-NAME treatment, or by a single exposure to immobilization stress for 2 hours followed by 24 hours of rest. In the hippocampus, the repeated treatment with L-NAME increased mRNA levels of AT1 receptors approximately 9-times compared to those in the control (untreated) group. Immobilization also increased AT1 receptor mRNA levels in the hippocampus which was similar to that induced by the L-NAME. The increase of AT1 receptor mRNA levels in the hippocampus of immobilized rats was not further altered when the animals were pretreated with L-NAME. In control rats, exposure to immobilization resulted in a significant rise in mRNA levels coding for AT1 receptors in the hypothalamus, but not in the thalamus. L-NAME treatment showed a tendency of increase in AT1 receptor mRNA levels in the hypothalamus. Moreover, when animals treated with L-NAME were subjected to immobilization, a further increase in AT1 receptor mRNA levels was observed in the hypothalamus in comparison with corresponding controls. The present data indicate that a single immobilization stress results in increased gene expression of AT1 receptors in the hypothalamus and hippocampus. The rise in AT1 mRNA levels in the same brain structures after repeated treatment with L-NAME allow to suggest an

  18. Evolution of a Double Amino Acid Substitution in the 5-Enolpyruvylshikimate-3-Phosphate Synthase in Eleusine indica Conferring High-Level Glyphosate Resistance1

    Science.gov (United States)

    Yu, Qin; Jalaludin, Adam; Han, Heping; Chen, Ming; Sammons, R. Douglas; Powles, Stephen B.

    2015-01-01

    Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I + P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action. PMID:25717039

  19. Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance.

    Science.gov (United States)

    Yu, Qin; Jalaludin, Adam; Han, Heping; Chen, Ming; Sammons, R Douglas; Powles, Stephen B

    2015-04-01

    Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I+P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action. © 2015 American Society of Plant Biologists. All Rights Reserved.

  20. Thymidylate synthase genetic polymorphism and plasma total homocysteine level in a group of Turkish patients with rheumatoid arthritis: relationship with disease activity and methotrexate toxicity.

    Science.gov (United States)

    Borman, Pınar; Taşbaş, Özgur; Karabulut, Halil; Tukun, Ajlan; Yorgancıoğlu, Rezan

    2015-01-01

    The polymorphism of thymidylate synthase (TS) gene and homocysteine are reported to have a relationship to methotrexate (MTX) metabolism, with conflicting results. The aim of this study was to determine homocysteine levels and the frequency of TS gene triple repeat (TS3R) and double repeat (TS2R) polymorphisms in a group of Turkish RA patients and evaluate its association with MTX toxicity and disease activity. Sixty-four patients with RA and 31 control subjects with a mean age of 48.7 ± 12.5 and 46.2 ± 13.4 years, were enrolled to the study. Demographic characteristics were obtained and number of patients with MTX-related adverse affects, were recorded in the patient group. The homocysteine levels and TS2R/TS3R polymorphisms of the TS gene were analyzed and the distribution of genotypes according to MTX toxicity and disease activity, were determined. The demographic properties were similar between the patient and control subjects. Folic acid supplementation with a mean dose of 5mg folic acid/week, was present in all patients. Thirty-six of the 64 patients showed adverse effects to MTX treatment. The frequency of TS2R and TS3R polymorphisms were found to be similar in the patient and control groups. TS2R and TS3R gene polymorphisms were found to be similar in patients with and without MTX-related adverse events. The mean homocysteine level was also similar in patients with and without TS gene polymorphism, but was found to be higher (12.45μmol/L vs 10.7μmol/L) in patients with MTX-related side effects than in patients without side effects. The mean level of homocysteine was correlated with levels of ESR in the patient group. In conclusion, homocysteine levels might effect the disease activity and toxicity of MTX but 2R and 3R polymorphisms in the TS gene, were not related with MTX-related toxicity in RA patients receiving folate supplementation. Further studies are needed to illuminate the polymorphisms in other enzymes that might be responsible from the MTX

  1. Investigation of the connection between the quality of protein, protein level and endogenous N excretion

    International Nuclear Information System (INIS)

    Koehler, R.; Gebhardt, G.

    1979-01-01

    The influence of various protein qualities as well as of different levels of protein on the amount of endogenous N excretion, metabolic fecal nitrogen (MFN) and endogenous urinary N (EUN) was determined in growing albino rats. The test rations were labelled with admixtures of 15 N-DL-methionine and 15 N-DL-lysine, respectively, or contained feed protein enriched with 15 N. EUN and MFN and their sum (the N maintenance requirement) showed the influence of the respective protein source and its dependence on the protein level. The endogenous N excretions showed an opposite tendency to the N balance; for high-quality protein feedstuffs with a high N balance (e.g. dried eggs) they are lower than for protein sources of inferior quality, with a low N-balance only (e.g. wheat gluten). Presumably this interaction of retention and maintenance is due to the complementary effect of exogenous and endogenous amino acids in the N and amino acid pool, respectively. Provided that the N dose and the live weight of the animals are comparable, the N balance appears to be more suitable as parameter for the description of the protein quality and the calculation of the protein utilisation than N retention, as the sum of N balance and the values of MFN and EUN (depending on the feedstuffs and the N level). (author)

  2. Human serum protein and C-reactive protein levels among HIV ...

    African Journals Online (AJOL)

    Human serum protein and C-reactive protein levels were determined among HIV patients visiting St Camillus Hospital, Uromi, Edo State, Nigeria, between January to March, 2013. Fifty (50) HIV patients (20 males; 30 females) and 50 control subjects (24 males; 26 females) were enrolled for this study. The clinical status of ...

  3. Inhibition of Glycogen Synthase Kinase or the Apoptotic Protein p53 Lowers the Threshold of Helium Cardioprotection In Vivo: The Role of Mitochondrial Permeability Transition

    Science.gov (United States)

    Pagel, Paul S.; Krolikowski, John G.; Pratt, Phillip F.; Shim, Yon Hee; Amour, Julien; Warltier, David C.; Weihrauch, Dorothee

    2008-01-01

    BACKGROUND Prosurvival signaling kinases inhibit glycogen synthase kinase-3β (GSK-3β) activity and stimulate apoptotic protein p53 degradation. Helium produces cardioprotection by activating prosurvival kinases, but whether GSK and p53 inhibition mediate this process is unknown. We tested the hypothesis that inhibition of GSK or p53 lowers the threshold of helium cardioprotection via a mitochondrial permeability transition pore (mPTP)-dependent mechanism. METHODS Rabbits (n = 85) instrumented for hemodynamic measurement and subjected to a 30 min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion received 0.9% saline (control), or 1, 3, or 5 cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture (fraction of inspired oxygen concentration = 0.30) before LAD occlusion. Other rabbits received the GSK inhibitor SB 216763 (SB21; 0.2 or 0.6 mg/kg), the p53 inhibitor pifithrin-α (PIF; 1.5 or 3.0 mg/kg), or SB21 (0.2 mg/kg) or PIF (1.5 mg/kg) plus helium (1 cycle) before LAD occlusion in the presence or absence of the mPTP opener atractyloside (5 mg/kg). RESULTS Helium reduced (P < 0.05) myocardial infarct size (35 ± 6 [n = 7], 25 ± 4 [n = 7], and 20 ± 3% [n = 6] of area at risk, 1, 3, and 5 cycles, respectively) compared with control (44 ± 6% [n = 7]). SB21 (0.6 [n = 7] but not 0.2 mg/kg [n = 6]) and PIF (3.0 [n = 6] but not 1.5 mg/kg [n = 7]) also reduced necrosis. SB21 (0.2 mg/kg) or 1.5 mg/kg PIF (1.5 mg/kg) plus helium (1 cycle; n = 6 per group) decreased infarct size to an equivalent degree as three cycles of helium alone, and this cardioprotection was blocked by atractyloside (n = 7 per group). CONCLUSIONS Inhibition of GSK or p53 lowers the threshold of helium-induced preconditioning via a mPTP-dependent mechanism in vivo. PMID:18713881

  4. The calcium-dependent protein kinase RcCDPK2 phosphorylates sucrose synthase at Ser11 in developing castor oil seeds.

    Science.gov (United States)

    Fedosejevs, Eric T; Gerdis, Suzanne A; Ying, Sheng; Pyc, Michal; Anderson, Erin M; Snedden, Wayne A; Mullen, Robert T; She, Yi-Min; Plaxton, William C

    2016-10-15

    Imported sucrose is cleaved by sucrose synthase (SUS) as a critical initial reaction in the biosynthesis of storage end-products by developing seeds. Although SUS is phosphorylated at a conserved seryl residue by an apparent CDPK (Ca 2+ -dependent protein kinase) in diverse plant tissues, the functions and mechanistic details of this process remain obscure. Thus, the native CDPK that phosphorylates RcSUS1 (Ricinus communis SUS1) at Ser 11 in developing COS (castor oil seeds) was highly purified and identified as RcCDPK2 by MS/MS. Purified RcSUS1-K (-kinase) and heterologously expressed RcCDPK2 catalyzed Ca 2+ -dependent Ser 11 phosphorylation of RcSUS1 and its corresponding dephosphopeptide, while exhibiting a high affinity for free Ca 2+ ions [K 0.5 (Ca 2+ ) < 0.4 µM]. RcSUS1-K activity, RcCDPK2 expression, and RcSUS1 Ser 11 phosphorylation peaked during early COS development and then declined in parallel. The elimination of sucrose import via fruit excision triggered RcSUS1 dephosphorylation but did not alter RcSUS1-K activity, suggesting a link between sucrose signaling and posttranslational RcCDPK2 control. Both RcCDPK2-mCherry and RcSUS1-EYFP co-localized throughout the cytosol when transiently co-expressed in tobacco suspension cells, although RcCDPK2-mCherry was also partially localized to the nucleus. Subcellular fractionation revealed that ∼20% of RcSUS1-K activity associates with microsomal membranes in developing COS, as does RcSUS1. In contrast with RcCDPK1, which catalyzes inhibitory phosphorylation of COS bacterial-type phosphoenolpyruvate carboxylase at Ser 451 , RcCDPK2 exhibited broad substrate specificity, a wide pH-activity profile centered at pH 8.5, and insensitivity to metabolite effectors or thiol redox status. Our combined results indicate a possible link between cytosolic Ca 2+ -signaling and the control of photosynthate partitioning during COS development. © 2016 The Author(s); published by Portland Press Limited on behalf of the

  5. Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

    Science.gov (United States)

    Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

    2016-12-21

    Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

  6. High-level transient expression of recombinant protein in lettuce.

    Science.gov (United States)

    Joh, Lawrence D; Wroblewski, Tadeusz; Ewing, Nicholas N; VanderGheynst, Jean S

    2005-09-30

    Transient expression following agroinfiltration of plant tissue was investigated as a system for producing recombinant protein. As a model system, Agrobacterium tumefaciens containing the beta-glucuronidase (GUS) gene was vacuum infiltrated into lettuce leaf disks. Infiltration with a suspension of 10(9) colony forming units/mL followed by incubation for 72 h at 22 degrees C in continuous darkness produced a maximum of 0.16% GUS protein based on dry tissue or 1.1% GUS protein based on total soluble protein. This compares favorably to expression levels for commercially manufactured GUS protein from transgenic corn seeds. A. tumefaciens culture medium pH between 5.6 and 7.0 and surfactant concentrations lettuce to produce GUS protein more rapidly, but final levels did not exceed the GUS production in leaves incubated in continuous darkness after 72 h at 22 degrees C. The kinetics of GUS expression during incubation in continuous light and dark were represented well using a logistic model, with rate constants of 0.30 and 0.29/h, respectively. To semi-quantitatively measure the GUS expression in large numbers of leaf disks, a photometric enhancement of the standard histochemical staining method was developed. A linear relationship with an R2 value of 0.90 was determined between log10 (% leaf darkness) versus log10 (GUS activity). Although variability in expression level was observed, agroinfiltration appears to be a promising technology that could potentially be scaled up to produce high-value recombinant proteins in planta. Copyright 2005 Wiley Periodicals, Inc

  7. Association of plasma protein C levels and coronary artery disease ...

    African Journals Online (AJOL)

    Several studies have shown the risk factor causes of coronary heart disease. In this study we tested the hypothesis that plasma protein C level might be used as a biomarker for coronary heart disease and myocardial infarction. The study included 60 men that were classified into 3 groups according to clinical examination; ...

  8. Serum protein and enzyme levels in rats following administration of ...

    African Journals Online (AJOL)

    The effects of caffeinated and non-caffeinated paracetamol administration, with or without vitamins A and E supplementation on the protein and enzyme levels in Wistar albino rats were investigated using cafeinated paracetamol and paracetamol as caffeinated and non-caffeinated paracetamol respectively, and water ...

  9. Effect Of Different Protein Levels on The Performance Of Growing ...

    African Journals Online (AJOL)

    T4 was superior to other treatments in terms of feed to gain ratio, efficiency of feed utilization and reproductive performance. Snails in T1 with 10% CP failed to lay eggs and had the least developed reproductive system. A diet of 15-20% C. P is therefore recommended for growing snails. Key words: Protein levels, snails, ...

  10. Interactive effect of dietary protein level and zilpaterol hydrochloride ...

    African Journals Online (AJOL)

    Bonsmara type steers were used to determine the effect of dietary zilpaterol hydrochloride (ZH) in combination with different dietary crude protein (CP) levels (100, 120 and 140 g CP/kg) on growth performance and meat quality. Treatment groups (T) consisted of 12 steers each. T1 – 100 g CP/kg + 0.15 mg ZH/kg live weight ...

  11. Effect Of Crude Protein Levels And Follicle Stimulation On Egg ...

    African Journals Online (AJOL)

    Two groups received 16% crude protein (CP) level diets and the other two groups, 32%. One each of the two groups received follicle stimulation, induced by administration of Clomifene citrate (1.5mg/kg) via cathetered 5ml syringe through the 10week experimental period, with feed and water offered ad libitum.

  12. Effect of dietary crude protein level on the performance and ...

    African Journals Online (AJOL)

    ویرایه

    2013-06-26

    Jun 26, 2013 ... The effects of increasing dietary levels of crude protein (CP) on growth, feed intake, feed efficiency and nutrient apparent ... matter intake (DMI) than the kids fed with 10.5, 12.8, .... Food and Agriculture Organization. Database ...

  13. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    International Nuclear Information System (INIS)

    Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

    2014-01-01

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus

  14. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  15. The Cutoff Level for Urine Protein in Urine Immunofixation Electrophoresis.

    Science.gov (United States)

    Ellidag, Hamit Yasar; Curek, Gulten; Eren, Esin; Aydin, Ozgur; Yilmaz, Necat

    2015-01-01

    Immunofixation electrophoresis (IFE) maintains its importance in diagnosing monoclonal gammopathies. In particular, urine IFE detects free light chains (FLC) in urine samples even at low concentrations and offers higher sensitivity compared to serum electrophoresis and serum IFE. The aim of the present study was to determine the place and significance of quantitative urinary protein measurement before IFE in interpreting the results of subsequent IFE and to determine the most appropriate protein concentrations for the appearance of bands. The records of a total of 600 patients, who underwent screening for Bence Jones proteinuria using IFE on 24-hour urine, were retrospectively reviewed. Urine IFE was performed using Helena SAS-I and SAS-I devices. The total protein concentration in the urine was quantitatively determined by the Pyrogallol red method, and the urine albumin level was determined using the immunoturbidimetric method. These analyses were measured on an Olympus/Beckmann AU5800. The evaluation of IFE results revealed that 311 patients had normal results, 108 patients had monoclonal bands, five patients had biclonal bands, 28 had polyclonal bands, and 148 patients had various degrees of proteinuria. ROC curves were created in order to determine the most appropriate urinary protein and albumin levels to observe bands in IFE. Accordingly, urine baseline protein level (mg/dL) showed the highest AUC value (cutoff value: 19.4 mg/dL, sensitivity: 92%, specificity: 98.2%, AUC: 0.972). The present study showed that quantitative protein measurement before IFE eliminated the disadvantages associated with the IFE method and its interpretation.

  16. Proteins in Complementary Food: What Is the Healthiest Level?

    Directory of Open Access Journals (Sweden)

    О. K. Netrebenko

    2015-01-01

    Full Text Available Adequate protein consumption in infants is a heavily debated issue. First, it is related to the formation of a new scientific field — “Infant prerequisites of man’s wellness and illness,” which directly indicates that excessive intake of proteins during infancy has long-term consequences and greatly contributes to obesity and chronic infectious diseases in adults; second, it is related to new technologies, which improve the protein component of infant formulas and bring them at par with breast milk in terms of quality and quantity. High protein consumption is related to bottle feeding, because starter and further infant formulas are richer in protein than breast milk. Protein-rich menus trigger production of insulinogenic amino acids, insulin and the insulin-like growth factor (IGF-1. High IFTcombined with branched-chain amino acids (leucine, valine, isoleucine, threonine activates a set of signalling molecules (mTOR, which are responsible for integrating metabolic and immune response. Repeated activation of mTOR coupled with regular intake of high-protein infant formulas causes health issues in adulthood. Diseases like diabetes type 2, obesity, arterial hypertension, cancer (particularly prostatic cancer, are related to overactivation of the mTOR signalling molecule complex. Intensive consumption of milk in today’s world is the key mTOR activator contributing to an increased risk of lifestyle diseases and triggering the mechanism of their development. The progressing infant formula industry allows to cut protein levels in starter and further infant formulas down to 12 g/l and, respectively, lower the risk of non-infectious diseases in adulthood. 

  17. Far-infrared radiation acutely increases nitric oxide production by increasing Ca2+ mobilization and Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

    International Nuclear Information System (INIS)

    Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

    2013-01-01

    Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser 1179 phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser 1179 phosphorylation. •FIR increases intracellular Ca 2+ levels. •Thermo-sensitive TRPV Ca 2+ channels are unlikely to be involved in the FIR-mediated eNOS-Ser 1179 phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser 1179 ) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca 2+ levels. Treatment with KN-93, a selective inhibitor of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. This study suggests that FIR radiation increases NO

  18. Far-infrared radiation acutely increases nitric oxide production by increasing Ca{sup 2+} mobilization and Ca{sup 2+}/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jung-Hyun; Lee, Sangmi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Cho, Du-Hyong [Department of Neuroscience, School of Medicine, Konkuk University, Seoul 143-701 (Korea, Republic of); Park, Young Mi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Kang, Duk-Hee [Division of Nephrology, Department of Internal Medicine, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Jo, Inho, E-mail: inhojo@ewha.ac.kr [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of)

    2013-07-12

    Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser{sup 1179} phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser{sup 1179} phosphorylation. •FIR increases intracellular Ca{sup 2+} levels. •Thermo-sensitive TRPV Ca{sup 2+} channels are unlikely to be involved in the FIR-mediated eNOS-Ser{sup 1179} phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser{sup 1179}) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca{sup 2+} levels. Treatment with KN-93, a selective inhibitor of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. This

  19. Genetic and environmental influences of surfactant protein D serum levels

    DEFF Research Database (Denmark)

    Sørensen, Grith Lykke; Hjelmborg, Jacob v. B.; Kyvik, Kirsten Ohm

    2006-01-01

    in the NH(2)-terminal region (Met11Thr) of the mature protein is significantly associated with the serum SP-D levels. A classic twin study was performed on a twin population including 1,476 self-reported healthy adults. The serum SP-D levels increased with male sex, age, and smoking status. The intraclass...... defining the constitutional serum level of SP-D and determine the magnitude of the genetic contribution to serum SP-D in the adult population. Recent studies have demonstrated that serum SP-D concentrations in children are genetically determined and that a single nucleotide polymorphism (SNP) located...

  20. Genetic and environmental influences of surfactant protein D serum levels

    DEFF Research Database (Denmark)

    Sorensen, G.L.; Hjelmborg, J.V.; Kyvik, K.O.

    2006-01-01

    defining the constitutional serum level of SP-D and determine the magnitude of the genetic contribution to serum SP-D in the adult population. Recent studies have demonstrated that serum SP-D concentrations in children are genetically determined and that a single nucleotide polymorphism (SNP) located...... in the NH(2)-terminal region (Met11Thr) of the mature protein is significantly associated with the serum SP-D levels. A classic twin study was performed on a twin population including 1,476 self-reported healthy adults. The serum SP-D levels increased with male sex, age, and smoking status. The intraclass...

  1. Total proteins and protein fractions levels in pregnant rats subjected to whole-body gamma irradiation

    International Nuclear Information System (INIS)

    Mansour, M.A.; Roushdy, H.M.; Mazhar, F.M.; Abu-Gabal, H.A.

    1986-01-01

    A total number of 180 mature rats (120 females and 60 males) weighing from 120-140 g were used to study the effect of two doses (2 and 4 Gy) whole-body gamma irradiation on the level of total protein and protein fractions in serum of pregnant rats during the period of organogenesis. It was found that the levels of total protein, albumin and gamma globulins significantly decreased according to the doses of exposure. The levels of alpha and beta globulins significantly increased more in the serum of rats exposed to 2 Gy than in rats exposed to 4 Gy. The level of A/G ratio significantly decreased more in the serum of rats exposed to 2Gy than in those exposed to 4 Gy

  2. Low copper and high manganese levels in prion protein plaques

    Science.gov (United States)

    Johnson, Christopher J.; Gilbert, P.U.P.A.; Abrecth, Mike; Baldwin, Katherine L.; Russell, Robin E.; Pedersen, Joel A.; McKenzie, Debbie

    2013-01-01

    Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system.

  3. Effect of feeding different dietary protein levels on reproductive ...

    African Journals Online (AJOL)

    A feeding trial was conducted to evaluate effects of feeding different dietary protein levels on reproductive biology of African mud catfish under hapa system. Catfish fingerlings (mean body weight (4.50± 0.01g) and total length (8.0±0.2cm) were randomly stocked at 20 fish per hapa (1m3). Five experimental diets with crude ...

  4. Isolation and Characterization of Three New Monoterpene Synthases from Artemisia annua

    Science.gov (United States)

    Ruan, Ju-Xin; Li, Jian-Xu; Fang, Xin; Wang, Ling-Jian; Hu, Wen-Li; Chen, Xiao-Ya; Yang, Chang-Qing

    2016-01-01

    Artemisia annua, an annual herb used in traditional Chinese medicine, produces a wealth of monoterpenes and sesquiterpenes, including the well-known sesquiterpene lactone artemisinin, an active ingredient in the treatment for malaria. Here we report three new monoterpene synthases of A. annua. From a glandular trichome cDNA library, monoterpene synthases of AaTPS2, AaTPS5, and AaTPS6, were isolated and characterized. The recombinant proteins of AaTPS5 and AaTPS6 produced multiple products with camphene and 1,8-cineole as major products, respectively, and AaTPS2 produced a single product, β-myrcene. Although both Mg2+ and Mn2+ were able to support their catalytic activities, altered product spectrum was observed in the presence of Mn2+ for AaTPS2 and AaTPS5. Analysis of extracts of aerial tissues and root of A. annua with gas chromatography–mass spectrometry detected more than 20 monoterpenes, of which the three enzymes constituted more than 1/3 of the total. Mechanical wounding induced the expression of all three monoterpene synthase genes, and transcript levels of AaTPS5 and AaTPS6 were also elevated after treatments with phytohormones of methyl jasmonate, salicylic acid, and gibberellin, suggesting a role of these monoterpene synthases in plant–environment interactions. The three new monoterpene synthases reported here further our understanding of molecular basis of monoterpene biosynthesis and regulation in plant. PMID:27242840

  5. Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

    CSIR Research Space (South Africa)

    Becker, JVW

    2010-01-01

    Full Text Available ] for hybridization to an Operon oligonucleotide array, interrogating 7797 70-mer oligonucleotides which repre- sent 4585 unique genes. RNA was extracted from several untreated, unsynchronized P. falciparum 3D7 cultures and cDNA synthesized for construction of a... background- subtracted intensity of a spot exceeded the average back- ground intensity of all spots in that channel [31]. cDNA synthesis and array hybridization A reference design was employed for array hybridisation, utilising the URR pool described...

  6. Cloning and expression of pineapple sucrosephosphate synthase ...

    African Journals Online (AJOL)

    A 1132-base pairs (bp) polymerase-chain-reaction product of sucrose-phosphate synthase (SPS) (EC 2.3.1.14) from pineapple (Ananas comosus cv. Comte de paris) fruit was cloned and nominated as Ac- SPS1. The sequence encodes a putative 377 amino acids protein containing two serine conserved features that had ...

  7. Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium.

    Science.gov (United States)

    Jin, Zhehao; Kim, Jin-Hee; Park, Sang Un; Kim, Soo-Un

    2016-12-01

    Two cDNAs for indole-3-glycerol phosphate lyase homolog were cloned from Polygonum tinctorium. One encoded cytosolic indole synthase possibly in indigoid synthesis, whereas the other encoded a putative tryptophan synthase α-subunit. Indigo is an old natural blue dye produced by plants such as Polygonum tinctorium. Key step in plant indigoid biosynthesis is production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit (TSA) homologs, PtIGL-short and -long, were isolated by RACE PCR from P. tinctorium. The genome of the plant contained two genes coding for IGL. The short and the long forms, respectively, encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli ΔtnaA ΔtrpA mutant on tryptophan-depleted agar plate signifying production of free indole, and thus was named indole synthase gene (PtINS). The long form, either intact or without the transit peptide sequence, did not complement the mutant and was tentatively named PtTSA. PtTSA was delivered into chloroplast as predicted by 42-residue-long targeting sequence, whereas PtINS was localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to fivefolds higher transcript level than that of PtTSA, and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA was significantly enhanced in the plant. The results indicate participation of PtINS in indigoid production.

  8. Reduced ceramide synthase 2 activity causes progressive myoclonic epilepsy

    DEFF Research Database (Denmark)

    Mosbech, Mai-Britt; Olsen, Anne S B; Neess, Ditte

    2014-01-01

    between genes involved in SL metabolism and epilepsy. METHODS: We used quantitative real-time PCR, Western blotting, and enzymatic assays to determine the mRNA, protein, and activity levels of ceramide synthase 2 (CERS2) in fiibroblasts isolated from parental control subjects and from a patient diagnosed...... with progressive myoclonic epilepsy (PME). Mass spectrometry and fluorescence microscopy were used to examine the effects of reduced CERS2 activity on cellular lipid composition and plasma membrane functions. RESULTS: We identify a novel 27 kb heterozygous deletion including the CERS2 gene in a proband diagnosed...... with PME. Compared to parental controls, levels of CERS2 mRNA, protein, and activity were reduced by ˜50% in fibroblasts isolated from this proband, resulting in significantly reduced levels of ceramides and sphingomyelins containing the very long-chain fatty acids C24:0 and C26:0. The change in SL...

  9. Diffusible Signal Factor (DSF) Synthase RpfF of Xylella fastidiosa Is a Multifunction Protein Also Required for Response to DSF

    OpenAIRE

    Ionescu, Michael; Baccari, Clelia; Da Silva, Aline Maria; Garcia, Angelica; Yokota, Kenji; Lindow, Steven E.

    2013-01-01

    Xylella fastidiosa, like related Xanthomonas species, employs an Rpf cell-cell communication system consisting of a diffusible signal factor (DSF) synthase, RpfF, and a DSF sensor, RpfC, to coordinate expression of virulence genes. While phenotypes of a ΔrpfF strain in Xanthomonas campestris could be complemented by its own DSF, the DSF produced by X. fastidiosa (XfDSF) did not restore expression of the XfDSF-dependent genes hxfA and hxfB to a ΔrpfF strain of X. fastidiosa, suggesting that Rp...

  10. C-reactive protein levels in patients with aggressive periodontitis.

    Science.gov (United States)

    Salzberg, Trang N; Overstreet, Benjamin T; Rogers, Jeffrey D; Califano, Joseph V; Best, Al M; Schenkein, Harvey A

    2006-06-01

    Sera from patients with periodontal infections contain elevated levels of C-reactive protein (CRP) compared to periodontally healthy individuals. Most studies to date have included patients with chronic periodontitis, and few investigators have studied CRP levels in subjects with aggressive periodontitis (AgP). The purpose of this study was to determine the relative levels of serum CRP in AgP patients and periodontally healthy subjects and to examine patients' characteristics that might account for intergroup differences. Serum samples were collected from 93 patients with generalized AgP (GAgP), from 97 patients with localized AgP (LAgP), and from 91 healthy controls (non-periodontitis [NP]). Periodontal examination consisted of plaque index, gingival index, probing depth, bleeding index, and attachment loss measurements. Current smoking was assessed by determination of serum cotinine levels by enzyme-linked immunosorbent assay (ELISA), and serum CRP levels were determined using a high-sensitivity ELISA assay. The three groups were significantly different from one another (P periodontal and demographic variables and current smoking, both mean probing depth and periodontal diagnosis remained correlated with CRP levels. Patients with AgP have statistically significant elevations in serum CRP levels compared to subjects without periodontitis. Elevated CRP in these subjects might represent a contribution of periodontal infections to systemic inflammation in relatively young individuals.

  11. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

    Science.gov (United States)

    Schimpl, Flávia Camila; Kiyota, Eduardo; Mayer, Juliana Lischka Sampaio; Gonçalves, José Francisco de Carvalho; da Silva, José Ferreira; Mazzafera, Paulo

    2014-09-01

    Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. C-Reactive Protein Levels in the Brugada Syndrome

    Directory of Open Access Journals (Sweden)

    Aimé Bonny

    2011-01-01

    Full Text Available Background. Inflammation in the Brugada syndrome (BrS and its clinical implication have been little studied. Aims. To assess the level of inflammation in BrS patients. Methods. All studied BrS patients underwent blood samples drawn for C-reactive protein (CRP levels at admission, prior to any invasive intervention. Patients with a previous ICD placement were controlled to exclude those with a recent (<14 days shock. We divided subjects into symptomatic (syncope or aborted sudden death and asymptomatic groups. In a multivariable analysis, we adjusted for significant variables (age, CRP ≥ 2 mg/L. Results. Fifty-four subjects were studied (mean age 45 ± 13 years, 49 (91% male. Twenty (37% were symptomatic. Baseline characteristics were similar in both groups. Mean CRP level was 1,4 ± 0,9 mg/L in asymptomatic and 2,4 ± 1,4 mg/L in symptomatic groups (P = .003. In the multivariate model, CRP concentrations ≥ 2 mg/L remained an independent marker for being symptomatic (P = .018; 95% CI: 1.3 to 19.3. Conclusion. Inflammation seems to be more active in symptomatic BrS. C-reactive protein concentrations ≥ 2 mg/L might be associated with the previous symptoms in BrS. The value of inflammation as a risk factor of arrhythmic events in BrS needs to be studied.

  13. A novel noncovalent complex of chorismate mutase and DAHP synthase from Mycobacterium tuberculosis: protein purification, crystallization and X-ray diffraction analysis

    International Nuclear Information System (INIS)

    Ökvist, Mats; Sasso, Severin; Roderer, Kathrin; Kast, Peter; Krengel, Ute

    2009-01-01

    Two shikimate-pathway enzymes from M. tuberculosis, the intracellular chorismate mutase (MtCM) and DAHP synthase (MtDS), were produced recombinantly and purified. MtCM was crystallized alone and in complex with MtDS and analyzed by X-ray diffraction. Chorismate mutase catalyzes a key step in the shikimate-biosynthetic pathway and hence is an essential enzyme in bacteria, plants and fungi. Mycobacterium tuberculosis contains two chorismate mutases, a secreted and an intracellular one, the latter of which (MtCM; Rv0948c; 90 amino-acid residues; 10 kDa) is the subject of this work. Here are reported the gene expression, purification and crystallization of MtCM alone and of its complex with another shikimate-pathway enzyme, DAHP synthase (MtDS; Rv2178c; 472 amino-acid residues; 52 kDa), which has been shown to enhance the catalytic efficiency of MtCM. The MtCM–MtDS complex represents the first noncovalent enzyme complex from the common shikimate pathway to be structurally characterized. Soaking experiments with a transition-state analogue are also reported. The crystals of MtCM and the MtCM–MtDS complex diffracted to 1.6 and 2.1 Å resolution, respectively

  14. Levels of acute phase proteins remain stable after ischemic stroke

    Directory of Open Access Journals (Sweden)

    Paik Myunghee C

    2006-10-01

    Full Text Available Abstract Background Inflammation and inflammatory biomarkers play an important role in atherosclerosis and cardiovascular disease. Little information is available, however, on time course of serum markers of inflammation after stroke. Methods First ischemic stroke patients ≥40 years old had levels of high-sensitivity C-reactive protein (hsCRP, serum amyloid A (SAA, and fibrinogen measured in plasma samples drawn at 1, 2, 3, 7, 14, 21 and 28 days after stroke. Levels were log-transformed as needed, and parametric and non-parametric statistical tests were used to test for evidence of a trend in levels over time. Levels of hsCRP and SAA were also compared with levels in a comparable population of stroke-free participants. Results Mean age of participants with repeated measures (n = 21 was 65.6 ± 11.6 years, and 13 (61.9% were men, and 15 (71.4% were Hispanic. Approximately 75% of patients (n = 15 had mild strokes (NIH Stroke Scale score 0–5. There was no evidence of a time trend in levels of hsCRP, SAA, or fibrinogen for any of the markers during the 28 days of follow-up. Mean log(hsCRP was 1.67 ± 1.07 mg/L (median hsCRP 6.48 mg/L among stroke participants and 1.00 ± 1.18 mg/L (median 2.82 mg/L in a group of 1176 randomly selected stroke-free participants from the same community (p = 0.0252. Conclusion Levels of hsCRP are higher in stroke patients than in stroke-free subjects. Levels of inflammatory biomarkers associated with atherosclerosis, including hsCRP, appear to be stable for at least 28 days after first ischemic stroke.

  15. Effect of Crude Protein Levels in Concentrate and Concentrate Levels in Diet on Fermentation

    Directory of Open Access Journals (Sweden)

    Dinh Van Dung

    2014-06-01

    Full Text Available The effect of concentrate mixtures with crude protein (CP levels 10%, 13%, 16%, and 19% and diets with roughage to concentrate ratios 80:20, 60:40, 40:60, and 20:80 (w/w were determined on dry matter (DM and organic matter (OM digestibility, and fermentation metabolites using an in vitro fermentation technique. In vitro fermented attributes were measured after 4, 24, and 48 h of incubation respectively. The digestibility of DM and OM, and total volatile fatty acid (VFA increased whereas pH decreased with the increased amount of concentrate in the diet (p<0.001, however CP levels of concentrate did not have any influence on these attributes. Gas production reduced with increased CP levels, while it increased with increasing concentrate levels. Ammonia nitrogen (NH3-N concentration and microbial CP production increased significantly (p<0.05 by increasing CP levels and with increasing concentrate levels in diet as well, however, no significant difference was found between 16% and 19% CP levels. Therefore, 16% CP in concentrate and increasing proportion of concentrate up to 80% in diet all had improved digestibility of DM and organic matter, and higher microbial protein production, with improved fermentation characteristics.

  16. The muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise

    DEFF Research Database (Denmark)

    Aschenbach, W G; Suzuki, Y; Breeden, K

    2001-01-01

    In skeletal muscle both insulin and contractile activity are physiological stimuli for glycogen synthesis, which is thought to result in part from the dephosphorylation and activation of glycogen synthase (GS). PP1G/R(GL)(G(M)) is a glycogen/sarcoplasmic reticulum-associated type 1 phosphatase...... that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A. (2001) Mol....... Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of contraction...

  17. Sbg1 Is a Novel Regulator for the Localization of the β-Glucan Synthase Bgs1 in Fission Yeast.

    Directory of Open Access Journals (Sweden)

    Reshma Davidson

    Full Text Available Glucan synthases synthesize glucans, complex polysaccharides that are the major components in fungal cell walls and division septa. Studying regulation of glucan synthases is important as they are essential for fungal cell survival and thus popular targets for anti-fungal drugs. Linear 1,3-β-glucan is the main component of primary septum and is synthesized by the conserved β-glucan synthase Bgs1 in fission yeast cytokinesis. It is known that Rho1 GTPase regulates Bgs1 catalytic activity and the F-BAR protein Cdc15 plays a role in Bgs1 delivery to the plasma membrane. Here we characterize a novel protein Sbg1 that is present in a complex with Bgs1 and regulates its protein levels and localization. Sbg1 is essential for contractile-ring constriction and septum formation during cytokinesis. Sbg1 and Bgs1 physically interact and are interdependent for localization to the plasma membrane. Bgs1 is less stable and/or mis-targeted to vacuoles in sbg1 mutants. Moreover, Sbg1 plays an earlier and more important role in Bgs1 trafficking and localization than Cdc15. Together, our data reveal a new mode of regulation for the essential β-glucan synthase Bgs1 by the novel protein Sbg1.

  18. Honey bee protein atlas at organ-level resolution.

    Science.gov (United States)

    Chan, Queenie W T; Chan, Man Yi; Logan, Michelle; Fang, Yuan; Higo, Heather; Foster, Leonard J

    2013-11-01

    Genome sequencing has provided us with gene lists but cannot tell us where and how their encoded products work together to support life. Complex organisms rely on differential expression of subsets of genes/proteins in organs and tissues, and, in concert, evolved to their present state as they function together to improve an organism's overall reproductive fitness. Proteomics studies of individual organs help us understand their basic functions, but this reductionist approach misses the larger context of the whole organism. This problem could be circumvented if all the organs in an organism were comprehensively studied by the same methodology and analyzed together. Using honey bees (Apis mellifera L.) as a model system, we report here an initial whole proteome of a complex organism, measuring 29 different organ/tissue types among the three honey bee castes: queen, drone, and worker. The data reveal that, e.g., workers have a heightened capacity to deal with environmental toxins and queens have a far more robust pheromone detection system than their nestmates. The data also suggest that workers altruistically sacrifice not only their own reproductive capacity but also their immune potential in favor of their queen. Finally, organ-level resolution of protein expression offers a systematic insight into how organs may have developed.

  19. Serum transferrin levels in children with protein-energy malnutrition

    Directory of Open Access Journals (Sweden)

    Selime Aydoğdu

    2013-03-01

    Full Text Available Objective: Although the diagnosis of patients with severemalnutrition is easy, it is very difficult to recognize patientswith mild and moderate malnutrition. A variety of methodsattempts to develop for early diagnosis of these cases.In this study, we evaluated the serum transferrin and albuminlevels in children with mild, moderate and severeprotein-energy malnutrition (PEM.Materials and methods: Children admitted to our policlinic,aged between 3 and 25 months, 45 subjects withPEM and 39 healthy subjects (control group were evaluated.According to the Gomez, Waterlow and Kanawatisubjects with PEM were divided in 3 subgroups mild,moderate and severe PEM. Anthropometric measurementsand biochemical results of 4 groups were compared.Results: For albumin levels in mild to moderate PEMgroups, 37.7% sensitivity, and 28.5% specificity, positivepredictive value 54%; negative predictive value 16.6%was found. For severe PEM sensitivity, specificity, positivepredictive value and negative predictive value were71%, 62.5%, 45%, and 83.3% respectively.With respect to the levels of transferrin, a significant differencewas found between mild PEM-control and moderatePEM-control groups (p0.05.Conclusion: Our study results showed that albumin isa weak indicator in mild-moderate PEM. In these cases,serum transferrin level reduces before decreasing of albuminlevel, thus it may be an early sensitive finding thatcan be used as a sensitive parameter in the diagnosis ofearly stages of malnutrition.Key words: Protein energy malnutrition, children, albumin,transferrin

  20. F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis

    NARCIS (Netherlands)

    van der Laan, M; Bechtluft, P; Kol, S; Nouwen, N; Driessen, AJM

    2004-01-01

    The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a

  1. the effect of dietary energy and protein levels on the composition

    African Journals Online (AJOL)

    Zannel

    Keywords: Breeding ostriches, nutrition, energy, protein, amino acids, egg ... Yolk is an important nutritional component of the avian egg because .... 3 (energy) x 3 (protein) factorial design with energy and protein levels featuring as main factors. ... No significant interactions were observed between energy and protein levels.

  2. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B. (DuPont); (NWU)

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  3. Variability of protein level and phosphorylation status caused by biopsy protocol design in human skeletal muscle analyses

    Directory of Open Access Journals (Sweden)

    Caron Marc-André

    2011-11-01

    Full Text Available Abstract Background Bergström needle biopsy is widely used to sample skeletal muscle in order to study cell signaling directly in human tissue. Consequences of the biopsy protocol design on muscle protein quantity and quality remain unclear. The aim of the present study was to assess the impact of different events surrounding biopsy protocol on the stability of the Western blot signal of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1, Akt, glycogen synthase kinase-3β (GSK-3β, muscle RING finger protein 1 (MuRF1 and p70 S6 kinase (p70 S6K. Six healthy subjects underwent four biopsies of the vastus lateralis, distributed into two distinct visits spaced by 48 hrs. At visit 1, a basal biopsy in the right leg was performed in the morning (R1 followed by a second in the left leg in the afternoon (AF. At visit 2, a second basal biopsy (R2 was collected from the right leg. Low intensity mobilization (3 × 20 right leg extensions was performed and a final biopsy (Mob was collected using the same incision site as R2. Results Akt and p70 S6K phosphorylation levels were increased by 83% when AF biopsy was compared to R1. Mob condition induced important phosphorylation of p70 S6K when compared to R2. Comparison of R1 and R2 biopsies revealed a relative stability of the signal for both total and phosphorylated proteins. Conclusions This study highlights the importance to standardize muscle biopsy protocols in order to minimize the method-induced variation when analyzing Western blot signals.

  4. A novel noncovalent complex of chorismate mutase and DAHP synthase from Mycobacterium tuberculosis: protein purification, crystallization and X-ray diffraction analysis

    Science.gov (United States)

    Ökvist, Mats; Sasso, Severin; Roderer, Kathrin; Kast, Peter; Krengel, Ute

    2009-01-01

    Chorismate mutase catalyzes a key step in the shikimate-biosynthetic pathway and hence is an essential enzyme in bacteria, plants and fungi. Mycobacterium tuberculosis contains two chorismate mutases, a secreted and an intracellular one, the latter of which (MtCM; Rv0948c; 90 amino-acid residues; 10 kDa) is the subject of this work. Here are reported the gene expression, purification and crystallization of MtCM alone and of its complex with another shikimate-pathway enzyme, DAHP synthase (MtDS; Rv2178c; 472 amino-acid residues; 52 kDa), which has been shown to enhance the catalytic efficiency of MtCM. The MtCM–MtDS complex represents the first noncovalent enzyme complex from the common shikimate pathway to be structurally characterized. Soaking experiments with a transition-state analogue are also reported. The crystals of MtCM and the MtCM–MtDS complex diffracted to 1.6 and 2.1 Å resolution, respectively. PMID:19851019

  5. Localization of Nitric Oxide Synthase-containing Neurons in the Bat Visual Cortex and Co-localization with Calcium-binding Proteins

    International Nuclear Information System (INIS)

    Gu, Ya-Nan; Kim, Hang-Gu; Jeon, Chang-Jin

    2015-01-01

    Microchiroptera (microbats) is a suborder of bats thought to have degenerated vision. However, many recent studies have shown that they have visual ability. In this study, we labeled neuronal nitric oxide synthase (nNOS)—the synthesizing enzyme of the gaseous non-synaptic neurotransmitter nitric oxide—and co-localized it with calbindin D28K (CB), calretinin (CR), and parvalbumin (PV) in the visual cortex of the greater horseshoe bat (Rhinolophus ferrumequinum, a species of microbats). nNOS-immunoreactive (IR) neurons were found in all layers of the visual cortex. Intensely labeled neurons were most common in layer IV, and weakly labeled neurons were most common in layer VI. Majority of the nNOS-IR neurons were round- or oval-type neurons; no pyramidal-type neurons were found. None of these neurons co-localized with CB, CR, or PV. However, the synthesis of nitric oxide in the bat visual cortex by nNOS does not depend on CB, CR, or PV

  6. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  7. Effect of Eimeria acervulina infection on cell-specific xanthine oxidase (XO) and inducible NO synthase (iNOS) activities and duodenal protein tyrosine nitration (NTp) in chickens

    Science.gov (United States)

    Intracellular generation of nitric oxide (NO) and superoxide anion (O¯2) during pro-inflammatory stress can result in the formation of 3'-nitrotyrosine proteins (NTp) that correlate with alteration in protein function and metabolic impairment. Our objective was to determine the cell-specific relati...

  8. F-box only protein 2 (Fbxo2) regulates amyloid precursor protein levels and processing.

    Science.gov (United States)

    Atkin, Graham; Hunt, Jack; Minakawa, Eiko; Sharkey, Lisa; Tipper, Nathan; Tennant, William; Paulson, Henry L

    2014-03-07

    The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

  9. The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels

    International Nuclear Information System (INIS)

    King, Taj D.; Gandy, Johanna C.; Bijur, Gautam N.

    2006-01-01

    The ubiquitously expressed protein glycogen synthase kinase-3 (GSK3) is constitutively active, however its activity is markedly diminished following phosphorylation of Ser21 of GSK3α and Ser9 of GSK3β. Although several kinases are known to phosphorylate Ser21/9 of GSK3, for example Akt, relatively much less is known about the mechanisms that cause the dephosphorylation of GSK3 at Ser21/9. In the present study KCl-induced plasma membrane depolarization of SH-SY5Y cells, which increases intracellular calcium concentrations caused a transient decrease in the phosphorylation of Akt at Thr308 and Ser473, and GSK3 at Ser21/9. Overexpression of the selective protein phosphatase-1 inhibitor protein, inhibitor-2, increased basal GSK3 phosphorylation at Ser21/9 and significantly blocked the KCl-induced dephosphorylation of GSK3β, but not GSK3α. The phosphorylation of Akt was not affected by the overexpression of inhibitor-2. GSK3 activity is known to affect sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) levels. Overexpression of inhibitor-2 or treatment of cells with the GSK3 inhibitors lithium and SB216763 increased the levels of SERCA2. These results indicate that the protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation induced by KCl and that GSK3 activity regulates SERCA2 levels

  10. Gene-specific correlation of RNA and protein levels in human cells and tissues

    DEFF Research Database (Denmark)

    Edfors, Fredrik; Danielsson, Frida; Hallström, Björn M.

    2016-01-01

    An important issue for molecular biology is to establish whether transcript levels of a given gene can be used as proxies for the corresponding protein levels. Here, we have developed a targeted proteomics approach for a set of human non-secreted proteins based on parallel reaction monitoring...... to measure, at steady-state conditions, absolute protein copy numbers across human tissues and cell lines and compared these levels with the corresponding mRNA levels using transcriptomics. The study shows that the transcript and protein levels do not correlate well unless a gene-specific RNA-to-protein (RTP...

  11. Recent Advances in the Development of Mammalian Geranylgeranyl Diphosphate Synthase Inhibitors

    Directory of Open Access Journals (Sweden)

    Staci L. Haney

    2017-05-01

    Full Text Available The enzyme geranylgeranyl diphosphate synthase (GGDPS catalyzes the synthesis of the 20-carbon isoprenoid geranylgeranyl diphosphate (GGPP. GGPP is the isoprenoid donor for protein geranylgeranylation reactions catalyzed by the enzymes geranylgeranyl transferase (GGTase I and II. Inhibitors of GGDPS result in diminution of protein geranylgeranylation through depletion of cellular GGPP levels, and there has been interest in GGDPS inhibitors as potential anti-cancer agents. Here we discuss recent advances in the development of GGDPS inhibitors, including insights gained by structure-function relationships, and review the preclinical data that support the continued development of this novel class of drugs.

  12. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants.

    Science.gov (United States)

    Degenhardt, Jörg; Köllner, Tobias G; Gershenzon, Jonathan

    2009-01-01

    The multitude of terpene carbon skeletons in plants is formed by enzymes known as terpene synthases. This review covers the monoterpene and sesquiterpene synthases presenting an up-to-date list of enzymes reported and evidence for their ability to form multiple products. The reaction mechanisms of these enzyme classes are described, and information on how terpene synthase proteins mediate catalysis is summarized. Correlations between specific amino acid motifs and terpene synthase function are described, including an analysis of the relationships between active site sequence and cyclization type and a discussion of whether specific protein features might facilitate multiple product formation.

  13. Interactive effect of dietary protein level and zilpaterol hydrochloride ...

    African Journals Online (AJOL)

    p2492989

    feedlot performance and meat quality of steers ... result of decreased protein degradation and increased protein synthesis (Fiems, ... sheep as well as data from some BAA trials provide evidence to associate ... The perception is that meat is now ... Table 1 Dietary treatments of steers with a mean live weight of 278 ± 20 kg ...

  14. Effect of graded levels and sources of protein on scrotal ...

    African Journals Online (AJOL)

    Iso caloric rations (10.50 MJ/kg DM ME) were formulated using non-conventional protein source (maize offal and dry layer litter) to contain 12.11% CP, 14.96% CP, and 17.94% CP and fed to groups A, B and C respectively. Another ration was formulated using conventional protein source (maize, wheat bran, groundnut ...

  15. Exploring Sequence Characteristics Related to High- Level Production of Secreted Proteins in Aspergillus niger

    NARCIS (Netherlands)

    Van den Berg, B.A.; Reinders, M.J.T.; Hulsman, M.; Wu, L.; Pel, H.J.; Roubos, J.A.; De Ridder, D.

    2012-01-01

    Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large

  16. Studies on the Active Site of Deacetoxycephalosporin C Synthase

    NARCIS (Netherlands)

    Lloyd, Matthew D.; Lee, Hwei-Jen; Harlos, Karl; Zhang, Zhi-Hong; Baldwin, Jack E.; Schofield, Christopher J.; Charnock, John M.; Garner, C. David; Hara, Takane; Terwisscha van Scheltinga, Anke C.; Valegård, Karin; Viklund, Jenny A.C.; Hajdu, Janos; Andersson, Inger; Danielsson, Åke; Bhikhabhai, Rama

    1999-01-01

    The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25% of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of

  17. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    cells were quantitated using enzyme immunometric assays. The activity of GSK-3β (serine-9-phosphorylated GSK-3β/total GSK-3β) was lower at baseline compared with follow-up. No significant mean change over time was observed in levels of total GSK-3β and serine-9-phosphorylated GSK-3β. Exploratory......Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...... with emotional lability, subjective mood fluctuations and cognitive function in healthy individuals. Thirty-seven healthy subjects were evaluated with neuropsychological tests and blood samples at baseline and 12-week follow-up. Total GSK-3β and serine-9-phosphorylated GSK-3β in peripheral blood mononuclear...

  18. Coordination modes of tyrosinate-ligated catalase-type heme enzymes: magnetic circular dichroism studies of Plexaura homomalla allene oxide synthase, Mycobacterium avium ssp. paratuberculosis protein-2744c, and bovine liver catalase in their ferric and ferrous states.

    Science.gov (United States)

    Bandara, D M Indika; Sono, Masanori; Bruce, Grant S; Brash, Alan R; Dawson, John H

    2011-12-01

    Bovine liver catalase (BLC), catalase-related allene oxide synthase (cAOS) from Plexaura homomalla, and a recently isolated protein from the cattle pathogen Mycobacterium avium ssp. paratuberculosis (MAP-2744c (MAP)) are all tyrosinate-ligated heme enzymes whose crystal structures have been reported. cAOS and MAP have low (enzymes in their ferric and ferrous states using magnetic circular dichroism and UV-visible absorption spectroscopy. The MAP protein shows remarkable spectral similarities to cAOS and BLC in its native Fe(III) state, but clear differences from ferric proximal heme ligand His93Tyr Mb (myoglobin) mutant, which may be attributed to the presence of an Arg(+)-N(ω)-H···¯O-Tyr (proximal heme axial ligand) hydrogen bond in the first three heme proteins. Furthermore, the spectra of Fe(III)-CN¯, Fe(III)-NO, Fe(II)-NO (except for five-coordinate MAP), Fe(II)-CO, and Fe(II)-O(2) states of cAOS and MAP, but not H93Y Mb, are also similar to the corresponding six-coordinate complexes of BLC, suggesting that a tyrosinate (Tyr-O¯) is the heme axial ligand trans to the bound ligands in these complexes. The Arg(+)-N(ω)-H to ¯O-Tyr hydrogen bond would be expected to modulate the donor properties of the proximal tyrosinate oxyanion and, combined with the subtle differences in the catalytic site structures, affect the activities of cAOS, MAP and BLC. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  20. A Cyanobacterial Chlorophyll Synthase-HliD Complex Associates with the Ycf39 Protein and the YidC/Alb3 Insertase

    Czech Academy of Sciences Publication Activity Database

    Chidgey, Jack W.; Linhartová, Markéta; Komenda, Josef; Jackson, P. J.; Dickman, M. J.; Canniffe, D. P.; Koník, P.; Pilný, Jan; Hunter, C. N.; Sobotka, Roman

    2014-01-01

    Roč. 26, č. 2 (2014), s. 1267-1279 ISSN 1040-4651 R&D Projects: GA ČR P501/10/1000; GA MŠk ED2.1.00/03.0110 Institutional support: RVO:61388971 Keywords : Chlorophyll * Ycf39 protein * cyanobacteria Subject RIV: EE - Microbiology, Virology Impact factor: 9.338, year: 2014

  1. Role of Nitric Oxide, Nitric Oxide Synthase, Soluble Guanylyl Cyclase, and cGMP-Dependent Protein Kinase I in Mouse Stem Cell Cardiac Development

    Directory of Open Access Journals (Sweden)

    Valentina Spinelli

    2016-01-01

    Full Text Available Introduction and Aim. Nitric oxide (NO can trigger cardiac differentiation of embryonic stem cells (ESCs, indicating a cardiogenic function of the NO synthetizing enzyme(s (NOS. However, the involvement of the NO/NOS downstream effectors soluble guanylyl cyclase (sGC and cGMP activated protein kinase I (PKG-I is less defined. Therefore, we assess the involvement of the entire NO/NOS/sGC/PKG-I pathway during cardiac differentiation process. Methods. Mouse ESCs were differentiated toward cardiac lineages by hanging drop methodology for 21 days. NOS/sGC/PKG-I pathway was studied quantifying genes, proteins, enzymatic activities, and effects of inhibition during differentiation. Percentages of beating embryoid bodies (mEBs were evaluated as an index of cardiogenesis. Results and Discussion. Genes and protein expression of enzymes were increased during differentiation with distinctive kinetics and proteins possessed their enzymatic functions. Exogenous administered NO accelerated whereas the blockade of PKG-I strongly slowed cardiogenesis. sGC inhibition was effective only at early stages and NOS blockade ineffective. Of NOS/sGC/PKG-I pathway, PKG-I seems to play the prominent role in cardiac maturation. Conclusion. We concluded that exogenous administered NO and other pharmacological strategies able to increase the activity of PKG-I provide new tools to investigate and promote differentiation of cardiogenic precursors.

  2. Chromosomal localization of the human and mouse hyaluronan synthase genes

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, A.P.; McDonald, J.A. [Mayo Clinic Scottsdale, AZ (United States); Seldin, M.F. [Univ. of California Davis, CA (United States)] [and others

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  3. IDENTIFICATION AND CHARACTERIZATION OF THE SUCROSE SYNTHASE 2 GENE (Sus2 IN DURUM WHEAT

    Directory of Open Access Journals (Sweden)

    Mariateresa eVolpicella

    2016-03-01

    Full Text Available Sucrose transport is the central system for the allocation of carbon resources in vascular plants. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, represents a key enzyme in the control of the flow of carbon into starch biosynthesis. In the present study the genomic identification and characterization of the Sus2-2A and Sus2-2B genes coding for sucrose synthase in durum wheat (cultivars Ciccio and Svevo is reported. The genes were analyzed for their expression in different tissues and at different seed maturation stages, in four tetraploid wheat genotypes (Svevo, Ciccio, Primadur and 5-BIL42. The activity of the encoded proteins was evaluated by specific activity assays on endosperm extracts and their structure established by modelling approaches. The combined results of SUS2 expression and activity levels were then considered in the light of their possible involvement in starch yield.

  4. Isolation and characterization of three new monoterpene synthases from Artemisia annua

    Directory of Open Access Journals (Sweden)

    Ju-Xin eRuan

    2016-05-01

    Full Text Available Artemisia annua, an annual herb used in traditional Chinese medicine, produces a wealth of monoterpenes and sesquiterpenes, including the well-known sesquiterpene lactone artemisinin, an active ingredient in the treatment for malaria. Here we report three new monoterpene synthases of A. annua. From a glandular trichome cDNA library, monoterpene synthases of AaTPS2, AaTPS5 and AaTPS6, were isolated and characterized. The recombinant proteins of AaTPS5 and AaTPS6 produced multiple products with camphene and 1,8-cineole as major products, respectively, and AaTPS2 produced a single product, β-myrcene. Although both Mg2+ and Mn2+ were able to support their catalytic activities, altered product spectrum was observed in the presence of Mn2+ for AaTPS2 and AaTPS5. Analysis of extracts of aerial tissues and root of A. annua with gas chromatography-mass spectrometry (GC-MS detected more than 20 monoterpenes, of which the three enzymes constituted more than 1/3 of the total. Mechanical wounding induced the expression of all three monoterpene synthase genes, and transcript levels of AaTPS5 and AaTPS6 were also elevated after treatments with phytohormones of methyl jasmonate (MeJA, salicylic acid (SA and gibberellin (GA, suggesting a role of these monoterpene synthases in plant-environment interactions. The three new monoterpene synthases reported here further our understanding of molecular basis of monoterpene biosynthesis and regulation in plant.

  5. G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits

    NARCIS (Netherlands)

    Jimenez-Sainz, MC; Murga, C; Kavelaars, A; Jurado-Pueyo, M; Krakstad, BF; Heijnen, CJ; Mayor, F; Aragay, AM

    The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells

  6. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

    Science.gov (United States)

    Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E M; Jenkins, Jermaine L; Heimiller, Chelsea; Maines, Mahin D

    2016-08-01

    -Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation. © FASEB.

  7. Characterization of three chalcone synthase-like genes from apple (Malus x domestica Borkh.).

    Science.gov (United States)

    Yahyaa, Mosaab; Ali, Samah; Davidovich-Rikanati, Rachel; Ibdah, Muhammad; Shachtier, Alona; Eyal, Yoram; Lewinsohn, Efraim; Ibdah, Mwafaq

    2017-08-01

    Apple (Malus x domestica Brokh.) is a widely cultivated deciduous tree species of significant economic importance. Apple leaves accumulate high levels of flavonoids and dihydrochalcones, and their formation is dependent on enzymes of the chalcone synthase family. Three CHS genes were cloned from apple leaves and expressed in Escherichia coli. The encoded recombinant enzymes were purified and functionally characterized. In-vitro activity assays indicated that MdCHS1, MdCHS2 and MdCHS3 code for proteins exhibiting polyketide synthase activity that accepted either p-dihydrocoumaroyl-CoA, p-coumaroyl-CoA, or cinnamoyl-CoA as starter CoA substrates in the presence of malonyl-CoA, leading to production of phloretin, naringenin chalcone, and pinocembrin chalcone. MdCHS3 coded a chalcone-dihydrochalcone synthase enzyme with narrower substrate specificity than the previous ones. The apparent Km values of MdCHS3 for p-dihydrocoumaryl-CoA and p-coumaryl-CoA were both 5.0 μM. Expression analyses of MdCHS genes varied according to tissue type. MdCHS1, MdCHS2 and MdCHS3 expression levels were associated with the levels of phloretin accumulate in the respective tissues. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Oxidant stress evoked damage in rat hepatocyte leading to triggered nitric oxide synthase (NOS levels on long term consumption of aspartame

    Directory of Open Access Journals (Sweden)

    Iyaswamy Ashok

    2015-12-01

    Full Text Available This study investigates how long-term (40 mg/kg b.wt consumption of aspartame can alter the antioxidant status, stress pathway genes, and apoptotic changes in the liver of Wistar albino rats. Numerous controversial reports are available on the use of aspartame as it releases methanol as one of its metabolites during metabolism. To mimic the human methanol metabolism the methotrexate treated rats were included to study the aspartame effects. The aspartame treated methotrexate (MTX animals showed a marked significant increase in the superoxide dismutase (SOD, catalase (CAT, lipid peroxidation (LPO, and Glutathione peroxidase (GPx activity in the liver from control and MTX control animals, and showed a significant decrease in reduced glutathione (GSH and protein thiol in aspartame treated animals. The aspartame treated MTX animals showed a marked significant decrease in the body weight, brain, and liver weight. The aspartame treated MTX animals showed a marked increase in the inducible nitric oxide (iNOS, neuronal nitric oxide (nNOS, c-fos, Heat shock protein (Hsp 70 Tumour necrosis Factor (TNFα, caspase 8, c-jun N terminal kinases (JNK 3 and Nuclear factor kappa B (NFkB gene expression in the liver from control and MTX control animals. The aspartame treated MTX animals showed a marked increase in the c-fos, Hsp 70, iNOS Caspase 8, and JNK 3 protein expression in the liver from control and MTX control animals indicating the enhancement of stress and apoptosis. The aspartame treated MTX animals showed a streak of marked DNA fragmentation in the liver. On immunohistochemical analysis aspartame treated animals showed brown colored positive hepatocytes indicating the stress specific and apoptotic protein expression. Since aspartame consumption is on the rise among people, it is essential to create awareness regarding the usage of this artificial sweetener.

  9. Zinc finger protein 521 overexpression increased transcript levels of ...

    Indian Academy of Sciences (India)

    2016-02-12

    Feb 12, 2016 ... Zinc finger protein 521 is highly expressed in brain, neural stem cells and early progenitors of the human .... Membranes were blocked for 1 h with 10% skim milk and ..... fat-like development of white fat and thermogenesis.

  10. Serum total proteins and creatinine levels in experimental gambian ...

    African Journals Online (AJOL)

    Attempt was therefore made to evaluate the effect of two strains of Trypanosoma brucei gambiense on total proteins and other serum biochemical parameters using vervet monkeys as a model. The outcome of both strains in vervet monkeys was traumatic as the monkeys died from infection 12 – 15 weeks post infection while ...

  11. Plasma protein carbonyl levels and breast cancer risk

    Czech Academy of Sciences Publication Activity Database

    Rössner ml., Pavel; Terry, M. B.; Gammon, M. D.; Agrawal, M.; Zhang, F. F.; Ferris, J.S.; Teitelbaum, S. L.; Eng, S. M.; Gaudet, M. M.; Neugut, A. I.; Santella, R. M.

    2007-01-01

    Roč. 11, č. 5 (2007), s. 1138-1148 ISSN 1582-1838 Institutional research plan: CEZ:AV0Z50390512 Keywords : oxidative stress * protein carbonyl * breast cancer Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 6.807, year: 2007

  12. Serum total protein, albumin and globulin levels in Trypanosoma ...

    African Journals Online (AJOL)

    The effect of orally administered Scoparia dulcis on Trypanosoma brucei-induced changes in serum total protein, albumin and globulin were investigated in rabbits over a period of twenty eight days. Results obtained show that infection resulted in hyperproteinaemia, hyperglobulinaemia and hypoalbuminaemia. However ...

  13. (−)-EPICATECHIN IMPROVES MITOCHONDRIAL RELATED PROTEIN LEVELS AND AMELIORATES OXIDATIVE STRESS IN DYSTROPHIC DELTA SARCOGLYCAN NULL MOUSE STRIATED MUSCLE

    Science.gov (United States)

    Ramirez-Sanchez, Israel; De los Santos, Sergio; Gonzalez-Basurto, Silvia; Canto, Patricia; Mendoza-Lorenzo, Patricia; Palma-Flores, Carlos; Ceballos-Reyes, Guillermo; Villarreal, Francisco; Zentella-Dehesa, Alejandro; Coral-Vazquez, Ramon

    2014-01-01

    Muscular dystrophies (MD) are a group of heterogeneous genetic disorders characterized by progressive striated muscle wasting and degeneration. Although the genetic basis for many of these disorders has been identified, the exact mechanism for disease pathogenesis remains unclear. The presence of oxidative stress (OS) is known to contribute to the pathophysiology and severity of the MD. Mitochondrial dysfunction is observed in MD and likely represents an important determinant of increased OS. Experimental antioxidant therapies have been implemented with the aim of protecting against disease progression, but results from clinical trials have been disappointing. In this study, we explored the capacity of the cacao flavonoid (−)-epicatechin (Epi) to mitigate OS by acting as a positive regulator of mitochondrial structure/function endpoints and redox balance control systems in skeletal and cardiac muscles of dystrophic, δ-sarcoglycan (δ-SG) null mice. Wild type or δ-SG null 2.5 month old male mice were treated via oral gavage with either water (control animals) or Epi (1 mg/kg, twice/day) for 2 weeks. Results evidence a significant normalization of total protein carbonylation, recovery of reduced/oxidized glutathione (GSH/GSSG ratio) and enhanced superoxide dismutase 2, catalase and citrate synthase activities with Epi treatment. These effects were accompanied by increases in protein levels for thiolredoxin, glutathione peroxidase, superoxide dismutase 2, catalase and mitochondrial endpoints. Furthermore, we evidence decreases in heart and skeletal muscle fibrosis, accompanied with an improvement in skeletal muscle function with treatment. These results warrant the further investigation of Epi as a potential therapeutic agent to mitigate MD associated muscle degeneration. PMID:25284161

  14. SUMO-fusion, purification, and characterization of a (+)-zizaene synthase from Chrysopogon zizanioides

    International Nuclear Information System (INIS)

    Hartwig, S.; Frister, T.; Alemdar, S.; Li, Z.; Scheper, T.; Beutel, S.

    2015-01-01

    An uncharacterized plant cDNA coding for a polypeptide presumably having sesquiterpene synthase activity, was expressed in soluble and active form. Two expression strategies were evaluated in Escherichia coli. The enzyme was fused to a highly soluble SUMO domain, in addition to being produced in an unfused form by a cold-shock expression system. Yields up to ∼325 mg/L −1 were achieved in batch cultivations. The 6x-His-tagged enzyme was purified employing an Ni 2+ -IMAC-based procedure. Identity of the protein was established by Western Blot analysis as well as peptide mass fingerprinting. A molecular mass of 64 kDa and an isoelectric point of pI 4.95 were determined by 2D gel electrophoresis. Cleavage of the fusion domain was possible by digestion with specific SUMO protease. The synthase was active in Mg 2+ containing buffer and catalyzed the production of (+)-zizaene (syn. khusimene), a precursor of khusimol, from farnesyl diphosphate. Product identity was confirmed by GC–MS and comparison of retention indices. Enzyme kinetics were determined by measuring initial reaction rates for the product, using varying substrate concentrations. By assuming a Michaelis–Menten model, kinetic parameters of K M  = 1.111 μM (±0.113), v max  = 0.3245 μM min −1 (±0.0035), k cat  = 2.95 min −1 , as well as a catalytic efficiency k cat /K M  = 4.43 × 10 4  M −1 s −1 were calculated. Fusion to a SUMO moiety can substantially increase soluble expression levels of certain hard to express terpene synthases in E. coli. The kinetic data determined for the recombinant synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution. - Highlights: • Uncharacterized (+)-zizaene synthase from C. zizanoides was cloned and expressed. • Fusion to SUMO and cold-shock induction

  15. SUMO-fusion, purification, and characterization of a (+)-zizaene synthase from Chrysopogon zizanioides

    Energy Technology Data Exchange (ETDEWEB)

    Hartwig, S.; Frister, T.; Alemdar, S.; Li, Z.; Scheper, T.; Beutel, S., E-mail: beutel@iftc.uni-hannover.de

    2015-03-20

    An uncharacterized plant cDNA coding for a polypeptide presumably having sesquiterpene synthase activity, was expressed in soluble and active form. Two expression strategies were evaluated in Escherichia coli. The enzyme was fused to a highly soluble SUMO domain, in addition to being produced in an unfused form by a cold-shock expression system. Yields up to ∼325 mg/L{sup −1} were achieved in batch cultivations. The 6x-His-tagged enzyme was purified employing an Ni{sup 2+}-IMAC-based procedure. Identity of the protein was established by Western Blot analysis as well as peptide mass fingerprinting. A molecular mass of 64 kDa and an isoelectric point of pI 4.95 were determined by 2D gel electrophoresis. Cleavage of the fusion domain was possible by digestion with specific SUMO protease. The synthase was active in Mg{sup 2+} containing buffer and catalyzed the production of (+)-zizaene (syn. khusimene), a precursor of khusimol, from farnesyl diphosphate. Product identity was confirmed by GC–MS and comparison of retention indices. Enzyme kinetics were determined by measuring initial reaction rates for the product, using varying substrate concentrations. By assuming a Michaelis–Menten model, kinetic parameters of K{sub M} = 1.111 μM (±0.113), v{sub max} = 0.3245 μM min{sup −1} (±0.0035), k{sub cat} = 2.95 min{sup −1}, as well as a catalytic efficiency k{sub cat}/K{sub M} = 4.43 × 10{sup 4} M{sup −1} s{sup −1} were calculated. Fusion to a SUMO moiety can substantially increase soluble expression levels of certain hard to express terpene synthases in E. coli. The kinetic data determined for the recombinant synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution. - Highlights: • Uncharacterized (+)-zizaene synthase from C. zizanoides was cloned

  16. Glycogen Synthase in Sertoli Cells: More Than Glycogenesis?

    Science.gov (United States)

    Maldonado, Rodrigo; Mancilla, Héctor; Villarroel-Espíndola, Franz; Slebe, Felipe; Slebe, Juan Carlos; Méndez, Raúl; Guinovart, Joan J; Concha, Ilona I

    2016-11-01

    Sertoli cell metabolism actively maintains the nutritional needs of germ cells. It has been described that after glucose incorporation in Sertoli cells, less than 1% is converted to glycogen suggesting low levels of glycogen synthase activity. Phosphorylation of muscle glycogen synthase (MGS) at serine 640 (pS640MGS) decreases its activity, and this form of the enzyme was discovered as a non-ribosomal protein that modulates the translation of a subset of transcripts in HeLa cells. The aim of our study was to functionally characterize MGS in cultured Sertoli cells, as well as to explore this new feature related to RNA molecules. We detected MGS in the cytoplasm of Sertoli cells as well as in the nuclei. The activity rates of the enzyme were extremely low indicating that MGS is expressed but almost inactive. Protein targeting to glycogen (PTG) overexpression was performed to activate MGS by dephosphorylation. PTG induced glycogen synthesis massively, confirming that this enzyme is present but inactive. This finding correlates with high levels of pS640MGS, which were assayed by phosphatase treatment. To explore a putative new function for MGS in Sertoli cells, we performed RNA immunoprecipitation coupled to microarray studies. The results revealed that MGS co-immunoprecipitated with the several mRNAs and also rRNAs. These findings indicate that MGS is expressed Sertoli cells but in an inactive form, and also support a possibly novel feature of this metabolic enzyme associated with RNA-related molecules. J. Cell. Biochem. 117: 2597-2607, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Blood profiling of proteins and steroids during weight maintenance with manipulation of dietary protein level and glycaemic index

    DEFF Research Database (Denmark)

    Wang, Ping; Holst, Claus; Astrup, Arne

    2012-01-01

    ) blood biomarkers of dietary protein and GI levels during the weight-maintenance phase. Blood samples were collected at baseline, after 8 weeks of low-energy diet-induced weight loss and after a 6-month dietary intervention period from female continued weight losers (n 48) and weight regainers (n 48......), evenly selected from four dietary groups that varied in protein and GI levels. The blood concentrations of twenty-nine proteins and three steroid hormones were measured. The changes in analytes during weight maintenance largely correlated negatively with the changes during weight loss, with some...

  18. Glycogen synthase activation by sugars in isolated hepatocytes.

    Science.gov (United States)

    Ciudad, C J; Carabaza, A; Bosch, F; Gòmez I Foix, A M; Guinovart, J J

    1988-07-01

    We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.

  19. Effect of Dietary Protein Level on the Expression of Proteins in the Gastrointestinal Tract of Young Pigs.

    Science.gov (United States)

    Ma, Xianyong; Tian, Zhimei; Deng, Dun; Cui, Yiyan; Qiu, Yueqin

    2018-05-02

    The objective of this research is to investigate the effect of protein level on proteins expression in the gastrointestinal tract of young pigs. Eighteen piglets (Duroc × Landrace × Yorkshire) were weaned at 28 days of age and randomly assigned to three diets with 20%, 17%, and 14% CP level, and four essential amino acids, Lys, Met, Thr, and Trp, in three diets met the requirements of weaned piglets. The experimental period lasted 45 days. Compared with the control (20% CP level), the average daily feed intake, the average daily gain, and gain feed ratio of the 17% CP group did not decrease ( P > 0.05), but those of 14% CP group decreased ( P protein digestion and absorption, lipid or carbon digestion and absorption, etc. were up-regulated in 17% CP group, while most of them were down-regulated in 14% CP group. Amino acids metabolism of gastric, pancreatic secretion of duodenum or steroid hormone biosynthesis of jejunum was down-regulated in the 17% CP group, but the lipid metabolism was up-regulated in the 14% CP group. Six proteins were selected for identification by Western-blot, and their changes had the same trend as the proteomics results. The protein level decreased from 20% to 17%, the growth performance was not affected, while the nutrient digestion and absorption or the immune function were improved, which implied that 17% protein level maybe benefit for nutrients absorption of pigs.

  20. Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Cunha, Elizabeth S.; Kawahara, Rebeca [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Kadowaki, Marina K. [Universidade Estadual do Oeste do Parana, Cascavel, PR (Brazil); Amstalden, Hudson G.; Noleto, Guilhermina R.; Cadena, Silvia Maria S.C.; Winnischofer, Sheila M.B. [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Martinez, Glaucia R., E-mail: grmartinez@ufpr.br [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil)

    2012-09-10

    Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: Black-Right-Pointing-Pointer Melanogenesis stimulation by L-tyrosine+NH{sub 4}Cl in B16-F10 melanoma cells increases ROS levels. Black-Right-Pointing-Pointer Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. Black-Right-Pointing-Pointer Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. Black-Right-Pointing-Pointer RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.

  1. [BIOINFORMATIC SEARCH AND PHYLOGENETIC ANALYSIS OF THE CELLULOSE SYNTHASE GENES OF FLAX (LINUM USITATISSIMUM)].

    Science.gov (United States)

    Pydiura, N A; Bayer, G Ya; Galinousky, D V; Yemets, A I; Pirko, Ya V; Podvitski, T A; Anisimova, N V; Khotyleva, L V; Kilchevsky, A V; Blume, Ya B

    2015-01-01

    A bioinformatic search of sequences encoding cellulose synthase genes in the flax genome, and their comparison to dicots orthologs was carried out. The analysis revealed 32 cellulose synthase gene candidates, 16 of which are highly likely to encode cellulose synthases, and the remaining 16--cellulose synthase-like proteins (Csl). Phylogenetic analysis of gene products of cellulose synthase genes allowed distinguishing 6 groups of cellulose synthase genes of different classes: CesA1/10, CesA3, CesA4, CesA5/6/2/9, CesA7 and CesA8. Paralogous sequences within classes CesA1/10 and CesA5/6/2/9 which are associated with the primary cell wall formation are characterized by a greater similarity within these classes than orthologous sequences. Whereas the genes controlling the biosynthesis of secondary cell wall cellulose form distinct clades: CesA4, CesA7, and CesA8. The analysis of 16 identified flax cellulose synthase gene candidates shows the presence of at least 12 different cellulose synthase gene variants in flax genome which are represented in all six clades of cellulose synthase genes. Thus, at this point genes of all ten known cellulose synthase classes are identify in flax genome, but their correct classification requires additional research.

  2. Cloning and sequence analysis of putative type II fatty acid synthase ...

    Indian Academy of Sciences (India)

    Prakash

    Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L. ... acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, β-ketoacyl-ACP .... Helix II plays a dominant role in the interaction ... main distinguishing features of plant ACPs in plastids and ..... synthase component; J. Biol.

  3. The predictive nature of transcript expression levels on protein expression in adult human brain.

    Science.gov (United States)

    Bauernfeind, Amy L; Babbitt, Courtney C

    2017-04-24

    Next generation sequencing methods are the gold standard for evaluating expression of the transcriptome. When determining the biological implications of such studies, the assumption is often made that transcript expression levels correspond to protein levels in a meaningful way. However, the strength of the overall correlation between transcript and protein expression is inconsistent, particularly in brain samples. Following high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses of adult human brain samples, we compared the correlation in the expression of transcripts and proteins that support various biological processes, molecular functions, and that are located in different areas of the cell. Although most categories of transcripts have extremely weak predictive value for the expression of their associated proteins (R 2 values of < 10%), transcripts coding for protein kinases and membrane-associated proteins, including those that are part of receptors or ion transporters, are among those that are most predictive of downstream protein expression levels. The predictive value of transcript expression for corresponding proteins is variable in human brain samples, reflecting the complex regulation of protein expression. However, we found that transcriptomic analyses are appropriate for assessing the expression levels of certain classes of proteins, including those that modify proteins, such as kinases and phosphatases, regulate metabolic and synaptic activity, or are associated with a cellular membrane. These findings can be used to guide the interpretation of gene expression results from primate brain samples.

  4. Effect of the level of dietary protein on the utilization of alpha-ketoisocaproate for protein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Kang, C.W.; Tungsanga, K.; Walser, M.

    1986-04-01

    The efficiency of alpha-ketoisocaproate (KIC) as a dietary substitute for leucine in rats on varying protein intake was estimated by an isotopic method, previously shown to yield the same results as comparative growth experiments. /sup 14/C-KIC and /sup 3/H-leucine are injected orally. Six hours later the ratio, R, of /sup 14/C//sup 3/H in isolated proteins, divided by the same ratio in the injectate is measured. This ratio has been shown to be approximately equal to nutritional efficiency of KIC relative to leucine. As dietary protein increased from 6.3% to 48.3%, whole body protein R decreased from 0.515 +/- 0.045 to 0.299 +/- 0.016. Variations with protein intake were noted in R of protein isolated from individual organs. The magnitude of R in these organs varied two-fold, in the following sequence: brain greater than heart greater than or equal to skeletal muscle greater than or equal to salivary gland greater than or equal to kidney greater than liver. Whole body protein R could be confidently predicted (r2 = 0.992) from R in the protein of kidney and muscle. Thus, the nutritional efficiency of KIC as a dietary substitute for leucine in individual organs as well as in the whole animal is strongly dependent on the level of protein intake.

  5. Effect of the level of dietary protein on the utilization of alpha-ketoisocaproate for protein synthesis

    International Nuclear Information System (INIS)

    Kang, C.W.; Tungsanga, K.; Walser, M.

    1986-01-01

    The efficiency of alpha-ketoisocaproate (KIC) as a dietary substitute for leucine in rats on varying protein intake was estimated by an isotopic method, previously shown to yield the same results as comparative growth experiments. 14 C-KIC and 3 H-leucine are injected orally. Six hours later the ratio, R, of 14 C/ 3 H in isolated proteins, divided by the same ratio in the injectate is measured. This ratio has been shown to be approximately equal to nutritional efficiency of KIC relative to leucine. As dietary protein increased from 6.3% to 48.3%, whole body protein R decreased from 0.515 +/- 0.045 to 0.299 +/- 0.016. Variations with protein intake were noted in R of protein isolated from individual organs. The magnitude of R in these organs varied two-fold, in the following sequence: brain greater than heart greater than or equal to skeletal muscle greater than or equal to salivary gland greater than or equal to kidney greater than liver. Whole body protein R could be confidently predicted (r2 = 0.992) from R in the protein of kidney and muscle. Thus, the nutritional efficiency of KIC as a dietary substitute for leucine in individual organs as well as in the whole animal is strongly dependent on the level of protein intake

  6. Glycogen synthase kinase 3 alpha phosphorylates and regulates the osteogenic activity of Osterix.

    Science.gov (United States)

    Li, Hongyan; Jeong, Hyung Min; Choi, You Hee; Lee, Sung Ho; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl

    2013-05-10

    Osteoblast-specific transcription factor Osterix is a zinc-finger transcription factor that required for osteoblast differentiation and new bone formation. The function of Osterix can be modulated by post-translational modification. Glycogen synthase kinase 3 alpha (GSK3α) is a multifunctional serine/threonine protein kinase that plays a role in the Wnt signaling pathways and is implicated in the control of several regulatory proteins and transcription factors. In the present study, we investigated how GSK3α regulates Osterix during osteoblast differentiation. Wide type GSK3α up-regulated the protein level, protein stability and transcriptional activity of Osterix. These results suggest that GSK3α regulates osteogenic activity of Osterix. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Heritability and genetic basis of protein level variation in an outbred population.

    Science.gov (United States)

    Parts, Leopold; Liu, Yi-Chun; Tekkedil, Manu M; Steinmetz, Lars M; Caudy, Amy A; Fraser, Andrew G; Boone, Charles; Andrews, Brenda J; Rosebrock, Adam P

    2014-08-01

    The genetic basis of heritable traits has been studied for decades. Although recent mapping efforts have elucidated genetic determinants of transcript levels, mapping of protein abundance has lagged. Here, we analyze levels of 4084 GFP-tagged yeast proteins in the progeny of a cross between a laboratory and a wild strain using flow cytometry and high-content microscopy. The genotype of trans variants contributed little to protein level variation between individual cells but explained >50% of the variance in the population's average protein abundance for half of the GFP fusions tested. To map trans-acting factors responsible, we performed flow sorting and bulk segregant analysis of 25 proteins, finding a median of five protein quantitative trait loci (pQTLs) per GFP fusion. Further, we find that cis-acting variants predominate; the genotype of a gene and its surrounding region had a large effect on protein level six times more frequently than the rest of the genome combined. We present evidence for both shared and independent genetic control of transcript and protein abundance: More than half of the expression QTLs (eQTLs) contribute to changes in protein levels of regulated genes, but several pQTLs do not affect their cognate transcript levels. Allele replacements of genes known to underlie trans eQTL hotspots confirmed the correlation of effects on mRNA and protein levels. This study represents the first genome-scale measurement of genetic contribution to protein levels in single cells and populations, identifies more than a hundred trans pQTLs, and validates the propagation of effects associated with transcript variation to protein abundance. © 2014 Parts et al.; Published by Cold Spring Harbor Laboratory Press.

  8. Predicting protein folding pathways at the mesoscopic level based on native interactions between secondary structure elements

    Directory of Open Access Journals (Sweden)

    Sze Sing-Hoi

    2008-07-01

    Full Text Available Abstract Background Since experimental determination of protein folding pathways remains difficult, computational techniques are often used to simulate protein folding. Most current techniques to predict protein folding pathways are computationally intensive and are suitable only for small proteins. Results By assuming that the native structure of a protein is known and representing each intermediate conformation as a collection of fully folded structures in which each of them contains a set of interacting secondary structure elements, we show that it is possible to significantly reduce the conformation space while still being able to predict the most energetically favorable folding pathway of large proteins with hundreds of residues at the mesoscopic level, including the pig muscle phosphoglycerate kinase with 416 residues. The model is detailed enough to distinguish between different folding pathways of structurally very similar proteins, including the streptococcal protein G and the peptostreptococcal protein L. The model is also able to recognize the differences between the folding pathways of protein G and its two structurally similar variants NuG1 and NuG2, which are even harder to distinguish. We show that this strategy can produce accurate predictions on many other proteins with experimentally determined intermediate folding states. Conclusion Our technique is efficient enough to predict folding pathways for both large and small proteins at the mesoscopic level. Such a strategy is often the only feasible choice for large proteins. A software program implementing this strategy (SSFold is available at http://faculty.cs.tamu.edu/shsze/ssfold.

  9. Effects of dietary protein level on growth, health and physiological parameters in growing-furring mink

    DEFF Research Database (Denmark)

    Damgaard, Birthe Marie; Larsen, Peter F.; Clausen, Tove

    2012-01-01

    The aim of the study was to investigate the effects of the dietary protein level and the feeding strategy on growth, health and physiological blood and liver parameters in growing-furring male mink. Effects of dietary protein levels ranging from 22% of metabolizable energy (MEp) to experimental p...

  10. Effect of nonsteroidal antiinflammatory drugs on the C-reactive protein level in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Tarp, Simon; Bartels, Else M.; Bliddal, Henning

    2012-01-01

    To evaluate the effects of oral nonsteroidal antiinflammatory drugs (NSAIDs) on C-reactive protein (CRP) levels in rheumatoid arthritis (RA) patients, with a prespecified focus on the different NSAIDs.......To evaluate the effects of oral nonsteroidal antiinflammatory drugs (NSAIDs) on C-reactive protein (CRP) levels in rheumatoid arthritis (RA) patients, with a prespecified focus on the different NSAIDs....

  11. Effect of dietary protein and energy levels on the growth ...

    African Journals Online (AJOL)

    Dr. A.O. Ani

    While there were significant interactions (P< 0.05) between TBNO and enzyme levels on DM, ... ingredients like soya bean meal, groundnut meal, etc vis- à-vis their acute ... bacterial infection and sulfaquinoxaline drugs against coccidioses.

  12. Heterologous Gene Expression of N-Terminally Truncated Variants of LipPks1 Suggests a Functionally Critical Structural Motif in the N-terminus of Modular Polyketide Synthase

    DEFF Research Database (Denmark)

    Yuzawa, Satoshi; Bailey, Constance B.; Fujii, Tatsu A.

    2017-01-01

    Streptomyces-derived, Well-characterized modular, polyketide synthase (PKS). Using this enzyme as a model, we experimentally investigated the effects of alternative TSSs using a heterologous host, Streptomyces venezuelae. One of the TSSs employed boosted the protein level by 59-fold and the product yield by 23...

  13. Respon Pertumbuhan Ayam Lokal Pedaging terhadap Suplementasi Protein Isolasi Biji-bijian (PIB dan Perbedaan Level Protein Ransum

    Directory of Open Access Journals (Sweden)

    M. Aman Yaman

    2009-10-01

    Full Text Available The response of local meat chicken growth to supplementation of isolated grain protein and the difference in ration protein level ABSTRACT. A research which aims to determine the response of local meat chicken growth of protein supplementation with Isolation Grains Protein (IGB and the difference in ration protein level has been conducted in the Laboratory of Experimental Farm, Animal Husbandry Department, Faculty of Agriculture, Syiah Kuala University-Darussalam, Banda Aceh for 90 days. This study used a completely randomized design factorial with 2 factors, consisting of factors namely male gender (JJ and female (JB and the ration is a combination of factors and levels IGB in the ration, ie: treatment A: 17% protein and 0.4% IGB; treatment B 19% protein and 0.6% IGB and treatment C 21% protein and 0.8% IGB. Each combination consisted of 4 replications and each replication consists of 5 chickens. Parameters observed in the study were weight gain, achievement of final weight, consumption, conversion and efficiency of ration. DOC used a derivative result of selection of local meat chicken which are in the process of selection. Data acquired and processed by ANOVA. The results showed that supplementation of IGB and ration protein level difference was significantly effect (P <0.01 on weight gain, final weight, rate of consumption, conversion efficiency of rations and rations, but there is no interaction effect between sex and ration factors . The highest weight gain obtained in the male local chicken achieved by feeding a ration B (93.23 grams, while the hen rations achieved by providing treatment C (63.86 grams / week. The highest final body weight of male chicken on treatment B (1491.5 gram/90 days and hens in treatment C (1061.5 gram/90 days. However, the highest ration consumption in both male and female local chickens obtained from the ration A. Feed conversion value and the best feed efficiency obtained in treatment B for the treatment of

  14. Developmental changes in uncoupling protein 1 and F(1)-ATPase subunit levels in the golden hamster brown adipose tissue mitochondria as determined by electron microscopy in situ immunocytochemistry

    Czech Academy of Sciences Publication Activity Database

    Bednár, Jan; Soukup, Tomáš

    2003-01-01

    Roč. 22, - (2003), s. 477-486 ISSN 0231-5882 R&D Projects: GA ČR GA304/00/1653 Grant - others:NATO Research project(XX) 979876 Institutional research plan: CEZ:AV0Z5011922 Keywords : immunoelectron microscopy * uncoupling protein 1 * mitochondrial ATP synthase Subject RIV: ED - Physiology Impact factor: 0.794, year: 2003

  15. COPS5 (Jab1) protein increases β site processing of amyloid precursor protein and amyloid β peptide generation by stabilizing RanBP9 protein levels.

    Science.gov (United States)

    Wang, Hongjie; Dey, Debleena; Carrera, Ivan; Minond, Dmitriy; Bianchi, Elisabetta; Xu, Shaohua; Lakshmana, Madepalli K

    2013-09-13

    Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid β peptide (Aβ) in the brain is central to the pathogenesis of Alzheimer's disease (AD). Therefore, the identification of molecules that regulate Aβ generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aβ generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aβ levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aβ generation, followed by increased soluble APP-β (sAPP-β) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aβ generation, implying that endogenous COPS5 regulates Aβ generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aβ generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aβ generation by stabilizing RanBP9 protein levels.

  16. Lithium Impairs Kidney Development and Inhibits Glycogen Synthase Kinase-3β in Collecting Duct Principal Cells

    DEFF Research Database (Denmark)

    Kjærsgaard, Gitte; Madsen, Kirsten; Marcussen, Niels

    level significantly whereas total GSK-3β abundance was unaltered. Li+ treatment increased α-Smooth Muscle Actin (α-SMA) protein level significantly whereas E-cadherin expression was unaltered. In summary, Li+ treatment impairs postnatal development of the kidney cortex and outer medulla and increases pGSK......The postnatal rat kidney is highly susceptible to Lithium (Li+), which leads to significant tissue injury. We hypothesized that Li+ impairs development of the kidney through entry into epithelial cells of the distal nephron, inhibition of Glycogen Synthase Kinase-3β (GSK-3β) through phosphorylation...... on serine9 (pGSK-3β)and subsequent epithelial to mesenchymal dedifferentiation (EMT). GSK-3β immunoreactive protein was associated with collecting ducts in developing and adult human and rat kidney. Total GSK-3β protein abundance was stable in medulla while it decreased in cortex in the postnatal period...

  17. Multidrug resistance-associated protein-1 (MRP1 genetic variants, MRP1 protein levels and severity of COPD

    Directory of Open Access Journals (Sweden)

    Rutgers Bea

    2010-05-01

    Full Text Available Abstract Background Multidrug resistance-associated protein-1 (MRP1 protects against oxidative stress and toxic compounds generated by cigarette smoking, which is the main risk factor for chronic obstructive pulmonary disease (COPD. We have previously shown that single nucleotide polymorphisms (SNPs in MRP1 significantly associate with level of FEV1 in two independent population based cohorts. The aim of our study was to assess the associations of MRP1 SNPs with FEV1 level, MRP1 protein levels and inflammatory markers in bronchial biopsies and sputum of COPD patients. Methods Five SNPs (rs212093, rs4148382, rs504348, rs4781699, rs35621 in MRP1 were genotyped in 110 COPD patients. The effects of MRP1 SNPs were analyzed using linear regression models. Results One SNP, rs212093 was significantly associated with a higher FEV1 level and less airway wall inflammation. Another SNP, rs4148382 was significantly associated with a lower FEV1 level, higher number of inflammatory cells in induced sputum and with a higher MRP1 protein level in bronchial biopsies. Conclusions This is the first study linking MRP1 SNPs with lung function and inflammatory markers in COPD patients, suggesting a role of MRP1 SNPs in the severity of COPD in addition to their association with MRP1 protein level in bronchial biopsies.

  18. Trypanosoma cruzi: Serum levels of nitric oxide and expression of inducible nitric oxide synthase in myocardium and spleen of dogs in the acute stage of infection with metacyclic or blood trypomastigotes.

    Science.gov (United States)

    Vieira, Paula Melo de Abreu; Francisco, Amanda Fortes; de Souza, Sheler Martins; Malaquias, Luiz Cosme Cotta; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro; Veloso, Vanja Maria; de Lana, Marta; Tafuri, Washington Luiz; Carneiro, Cláudia Martins

    2009-01-01

    The participation of nitric oxide (NO) in the control of blood parasitemia and parasitism during the acute phase of infection in dogs inoculated with blood trypomastigotes (BT) or metacyclic trypomastigotes (MT group) of Berenice-78 Trypanosoma cruzi strain has been evaluated. Animals of the MT group (n=4) presented increased levels of serum NO throughout the infection when compared with the BT (n=4) or control (n=4) groups, and a delay in parasitemia peak compared with the BT group. In spleen fragments, tissue parasitism was not observed but the MT group presented larger areas associated with inducible NO synthase (iNOS) in relation to BT and control groups. Heart fragments of MT-infected animals exhibited comparatively low tissue parasitism and high iNOS expression, while animals of the BT group presented high inflammatory infiltrate, high tissue parasitism and low iNOS expression. These results indicate that the source of inoculum can interfere with the development of the acute phase of Chagas disease, and may also trigger a distinct parasite-host interaction during this phase.

  19. Selective decrease of components of the creatine kinase system and ATP synthase complex in chronic Chagas disease cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Priscila Camillo Teixeira

    2011-06-01

    Full Text Available BACKGROUND: Chronic Chagas disease cardiomyopathy (CCC is an inflammatory dilated cardiomyopathy with a worse prognosis than other cardiomyopathies. CCC occurs in 30 % of individuals infected with Trypanosoma cruzi, endemic in Latin America. Heart failure is associated with impaired energy metabolism, which may be correlated to contractile dysfunction. We thus analyzed the myocardial gene and protein expression, as well as activity, of key mitochondrial enzymes related to ATP production, in myocardial samples of end-stage CCC, idiopathic dilated (IDC and ischemic (IC cardiomyopathies. METHODOLOGY/PRINCIPAL FINDINGS: Myocardium homogenates from CCC (N=5, IC (N=5 and IDC (N=5 patients, as well as from heart donors (N=5 were analyzed for protein and mRNA expression of mitochondrial creatine kinase (CKMit and muscular creatine kinase (CKM and ATP synthase subunits aplha and beta by immunoblotting and by real-time RT-PCR. Total myocardial CK activity was also assessed. Protein levels of CKM and CK activity were reduced in all three cardiomyopathy groups. However, total CK activity, as well as ATP synthase alpha chain protein levels, were significantly lower in CCC samples than IC and IDC samples. CCC myocardium displayed selective reduction of protein levels and activity of enzymes crucial for maintaining cytoplasmic ATP levels. CONCLUSIONS/SIGNIFICANCE: The selective impairment of the CK system may be associated to the loss of inotropic reserve observed in CCC. Reduction of ATP synthase alpha levels is consistent with a decrease in myocardial ATP generation through oxidative phosphorylation. Together, these results suggest that the energetic deficit is more intense in the myocardium of CCC patients than in the other tested dilated cardiomyopathies.

  20. Protein Secondary Structures (α-helix and β-sheet) at a Cellular Level and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    International Nuclear Information System (INIS)

    Yu, P.

    2007-01-01

    Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the α-helix and β-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of β-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution (∼10 μm). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of α-helixes and β-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P <0.05) the percentage of α-helixes (from 47.1% to 36.1%: S-FTIR absorption intensity), increased the

  1. GLUCOSE AND TOTAL PROTEIN LEVEL IN LABORATORY RATS UNDER CONDITIONS OF SHORT-TERM FASTING

    Directory of Open Access Journals (Sweden)

    Damir Suljević

    2013-09-01

    Full Text Available Glucose level (UV enzymatic method and total protein level (Biuret method were measured in the blood samples of the rats exposed to short-term starvation. We found a statistically significant increase in the glucose level in experimental animals during starvation, which is also evident in males and females in the experimental group (p <0.05, while decrease in the total protein level was not statistically significant. During starvation, more significant weight loss was observed in females compared to males.Key words: glucose, total protein, serum, Rattus

  2. Effect of dietary crude protein level on the performance and ...

    African Journals Online (AJOL)

    ویرایه

    2013-06-26

    Jun 26, 2013 ... kids who were 86 ± 3 days old with live weight of 9 ± 03 kg were used in a completely randomized design. ... matter intake (DMI) than the kids fed with 10.5, 12.8, .... black goats fed with 18% CP level with 20, 16 and 14%.

  3. Sublethal Effects of Diesel on Total Protein Levels and Cholesterol ...

    African Journals Online (AJOL)

    Michael Horsfall

    were handpicked at the Eagle Cement area of the New Calabar River and subjected to different levels ... groups when compared to the control in both the muscle and the viscera of the periwinkle. ... In Nigeria, oil industry operations are both offshore and onshore. ... This acts as a means of assessing the hazard or potential ...

  4. High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

    Science.gov (United States)

    Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

    2018-02-01

    Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Influence of protein level and supplemental methionine in practical rations for young endangered masked bobwhite quail

    Science.gov (United States)

    Serafin, J.A.

    1982-01-01

    A study was conducted to examine the protein requirement of young endangered masked Bobwhite quail (Colinus virginianus ridgwayi). Five practical starting rations containing 24 to 32% protein were fed alone and supplemented with methionine for 5 weeks. Supplemental methionine significantly improved growth of quail fed diets containing 24 and 26% protein. Increasing the protein level improved growth of quail fed unsupplemented diets but did not do so when diets contained supplemental methionine. A methionine-supplemented ration containing 24% protein appeared adequate for supporting rapid growth of masked Bobwhite quail.

  6. Predicting the catalytic sites of isopenicillin N synthase (IPNS ...

    African Journals Online (AJOL)

    Isopenicillin N synthase (IPNS) related Non-haem iron-dependent oxygenases and oxidases (NHIDOX) demonstrated a striking structural conservativeness, even with low protein sequence homology. It is evident that these enzymes have an architecturally similar catalytic centre with active ligands lining the reactive pocket.

  7. Isolation and expression of the Pneumocystis carinii thymidylate synthase gene

    DEFF Research Database (Denmark)

    Edman, U; Edman, J C; Lundgren, B

    1989-01-01

    The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar...

  8. Analysis of genetic variation of inducible nitric oxide synthase and ...

    African Journals Online (AJOL)

    The genetic diversity of 100 Malaysian native chickens was investigated using polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) for two candidate genes: inducible nitric oxide synthase (INOS) and natural resistance-associated macrophage protein 1 (NRAMP1). The two genes were selected ...

  9. Characterising the cellulose synthase complexes of cell walls

    NARCIS (Netherlands)

    Mansoori Zangir, N.

    2012-01-01

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as

  10. Interleukin-2-induced survival of natural killer (NK) cells involving phosphatidylinositol-3 kinase-dependent reduction of ceramide through acid sphingomyelinase, sphingomyelin synthase, and glucosylceramide synthase.

    Science.gov (United States)

    Taguchi, Yoshimitsu; Kondo, Tadakazu; Watanabe, Mitsumasa; Miyaji, Michihiko; Umehara, Hisanori; Kozutsumi, Yasunori; Okazaki, Toshiro

    2004-11-15

    Interleukin 2 (IL-2) rescued human natural killer (NK) KHYG-1 cells from apoptosis along with a reduction of ceramide. Conversely, an increase of ceramide inhibited IL-2-rescued survival. IL-2 deprivation-induced activation of acid sphingomyelinase (SMase) and inhibition of glucosylceramide synthase (GCS) and sphingomyelin synthase (SMS) were normalized by IL-2 supplementation. A phosphatidyl inositol-3 (PI-3) kinase inhibitor, LY294002, inhibited IL-2-rescued survival, but a mitogen-activated protein kinase inhibitor, PD98059, and an inhibitor of Janus tyrosine kinase/signal transducer and activator of transcription pathway, AG490, did not. LY294002 inhibited IL-2-induced reduction of ceramide through activation of acid SMase and inhibition of GCS and SMS, suggesting the positive involvement of PI-3 kinase in ceramide reduction through enzymatic regulation. Indeed, a constitutively active PI-3 kinase enhanced growth rate and ceramide reduction through inhibition of acid SMase and activation of GCS and SMS. Further, LY294002 inhibited IL-2-induced changes of transcriptional level as well as mRNA and protein levels in acid SMase and GCS but did not affect the stability of the mRNAs. These results suggest that PI-3 kinase-dependent reduction of ceramide through regulation of acid SMase, GCS, and SMS plays a role in IL-2-rescued survival of NK cells.

  11. Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase

    Science.gov (United States)

    Ober, Dietrich; Hartmann, Thomas

    1999-01-01

    Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids. PMID:10611289

  12. The effect of serum magnesium levels and serum endothelin-1 levels on bone mineral density in protein energy malnutrition.

    Science.gov (United States)

    Ozturk, C F; Karakelleoglu, C; Orbak, Z; Yildiz, L

    2012-06-01

    An inadequate and imbalanced intake of protein and energy results in protein-energy malnutrition (PEM). It is known that bone mineral density and serum magnesium levels are low in malnourished children. However, the roles of serum magnesium and endothelin-1 (ET-1) levels in the pathophysiology of bone mineralization are obscure. Thus, the relationships between serum magnesium and ET-1 levels and the changes in bone mineral density were investigated in this study. There was a total of 32 subjects, 25 of them had PEM and seven were controls. While mean serum ET-1 levels of the children with kwashiorkor and marasmus showed no statistically significant difference, mean serum ET-1 levels of both groups were significantly higher than that of the control group. Serum magnesium levels were lower than normal value in 9 (36%) of 25 malnourished children. Malnourished children included in this study were divided into two subgroups according to their serum magnesium levels. While mean serum ET-1 levels in the group with low magnesium levels were significantly higher than that of the group with normal magnesium levels (p malnutrition. Our study suggested that lower magnesium levels and higher ET-1 levels might be important factors in changes of bone mineral density in malnutrition. We recommend that the malnourished patients, especially with hypomagnesaemia, should be treated with magnesium early.

  13. Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii

    KAUST Repository

    Salunke, Rahul

    2018-05-14

    The mitochondrial F-type ATP synthase, a multi-subunit nanomotor, is critical for maintaining cellular ATP levels. In Toxoplasma gondii and other apicomplexan parasites, many subunit components, necessary for proper assembly and functioning of this enzyme, appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomer (~600 kDa) and dimer (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits, a, b and d, can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex will facilitate the development of novel anti-parasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.

  14. Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii

    KAUST Repository

    Salunke, Rahul; Mourier, Tobias; Banerjee, Manidipa; Pain, Arnab; Shanmugam, Dhanasekaran

    2018-01-01

    The mitochondrial F-type ATP synthase, a multi-subunit nanomotor, is critical for maintaining cellular ATP levels. In Toxoplasma gondii and other apicomplexan parasites, many subunit components, necessary for proper assembly and functioning of this enzyme, appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomer (~600 kDa) and dimer (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits, a, b and d, can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex will facilitate the development of novel anti-parasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.

  15. Synergistic Control of Kinetochore Protein Levels by Psh1 and Ubr2.

    Directory of Open Access Journals (Sweden)

    Eva Herrero

    2016-02-01

    Full Text Available The accurate segregation of chromosomes during cell division is achieved by attachment of chromosomes to the mitotic spindle via the kinetochore, a large multi-protein complex that assembles on centromeres. The budding yeast kinetochore comprises more than 60 different proteins. Although the structure and function of many of these proteins has been investigated, we have little understanding of the steady state regulation of kinetochores. The primary model of kinetochore homeostasis suggests that kinetochores assemble hierarchically from the centromeric DNA via the inclusion of a centromere-specific histone into chromatin. We tested this model by trying to perturb kinetochore protein levels by overexpressing an outer kinetochore gene, MTW1. This increase in protein failed to change protein recruitment, consistent with the hierarchical assembly model. However, we find that deletion of Psh1, a key ubiquitin ligase that is known to restrict inner kinetochore protein loading, does not increase levels of outer kinetochore proteins, thus breaking the normal kinetochore stoichiometry. This perturbation leads to chromosome segregation defects, which can be partially suppressed by mutation of Ubr2, a second ubiquitin ligase that normally restricts protein levels at the outer kinetochore. Together these data show that Psh1 and Ubr2 synergistically control the amount of proteins at the kinetochore.

  16. Association between protein C levels and mortality in patients with advanced prostate, lung and pancreatic cancer.

    Science.gov (United States)

    Wilts, I T; Hutten, B A; Meijers, J C M; Spek, C A; Büller, H R; Kamphuisen, P W

    2017-06-01

    Procoagulant factors promote cancer progression and metastasis. Protein C is involved in hemostasis, inflammation and signal transduction, and has a protective effect on the endothelial barrier. In mice, administration of activated protein C reduced experimental metastasis. We assessed the association between protein C and mortality in patients with three types of cancer. The study population consisted of patients with advanced prostate, non-small cell lung or pancreatic cancer, who participated in the INPACT trial (NCT00312013). The trial evaluated the addition of nadroparin to chemotherapy in patients with advanced malignancy. Patients were divided into tertiles based on protein C at baseline. The association between protein C levels and mortality was evaluated with Cox proportional hazard models. We analysed 477 patients (protein C tertiles: C level was 107% (IQR 92-129). In the lowest tertile, 75 patients per 100 patient-years died, as compared to 60 and 54 in the middle and high tertile, respectively. Lower levels of protein C were associated with increased mortality (in tertiles: HR for trend 1.18, 95%CI 1.02-1.36, adjusted for age, sex and nadroparin use; as a continuous variable: HR 1.004, 95%CI 1.00-1.008, p=0.07). Protein C seems inversely associated with mortality in patients with advanced prostate, lung and pancreatic cancer. Further research should validate protein C as a biomarker for mortality, and explore the effects of protein C on progression of cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Protein levels and colony development of Africanized and European honey bees fed natural and artificial diets.

    Science.gov (United States)

    Morais, M M; Turcatto, A P; Pereira, R A; Francoy, T M; Guidugli-Lazzarini, K R; Gonçalves, L S; de Almeida, J M V; Ellis, J D; De Jong, D

    2013-12-19

    Pollen substitute diets are a valuable resource for maintaining strong and health honey bee colonies. Specific diets may be useful in one region or country and inadequate or economically unviable in others. We compared two artificial protein diets that had been formulated from locally-available ingredients in Brazil with bee bread and a non-protein sucrose diet. Groups of 100 newly-emerged, adult workers of Africanized honey bees in Brazil and European honey bees in the USA were confined in small cages and fed on one of four diets for seven days. The artificial diets included a high protein diet made of soy milk powder and albumin, and a lower protein level diet consisting of soy milk powder, brewer's yeast and rice bran. The initial protein levels in newly emerged bees were approximately 18-21 µg/µL hemolymph. After feeding on the diets for seven days, the protein levels in the hemolymph were similar among the protein diet groups (~37-49 µg/µL after seven days), although Africanized bees acquired higher protein levels, increasing 145 and 100% on diets D1 and D2, respectively, versus 83 and 60% in the European bees. All the protein diets resulted in significantly higher levels of protein than sucrose solution alone. In the field, the two pollen substitute diets were tested during periods of low pollen availability in the field in two regions of Brazil. Food consumption, population development, colony weight, and honey production were evaluated to determine the impact of the diets on colony strength parameters. The colonies fed artificial diets had a significant improvement in all parameters, while control colonies dwindled during the dearth period. We conclude that these two artificial protein diets have good potential as pollen substitutes during dearth periods and that Africanized bees more efficiently utilize artificial protein diets than do European honey bees.

  18. Development, Characterization, and Optimization of Protein Level in Date Bars Using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Muhammad Nadeem

    2012-01-01

    Full Text Available This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM. Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD with 2 variables and 3 levels for each variable. Date bars were then analyzed for nutritional profile. Proximate composition revealed that addition of whey protein concentrate and vetch protein isolates improved the nutritional profile of date bars. Protein level, texture, and taste were considerably improved by incorporating 6.05% whey protein concentrate and 4.35% vetch protein isolates in date bar without affecting any sensory characteristics during storage. Response surface methodology was observed as an economical and effective tool to optimize the ingredient level and to discriminate the interactive effects of independent variables.

  19. human serum protein and c-reactive protein levels among hiv ...

    African Journals Online (AJOL)

    2016-09-30

    Sep 30, 2016 ... inflammation used to monitor HIV infection (Pepys and Hirschfield, 2003; Baker et al., 2010; Funderburg et al., 2010;. Neuhaus et ... from microbial infections, the CRP concentration can rise up to 300mg/L in 12-24 hours (Le Carrer et al., 1995; Vaishnavi,. 1996 ..... (pentaxins) and serum amyloid A protein.

  20. Animal Protein intake and the Effect of age on the level of Protein ...

    African Journals Online (AJOL)

    Some of the items such as eggs, milk and poultry meat were not consumed in reasonable quantities due to poor economic situation of the family. The results suggested that except for the fathers, other groups such as mothers and children were consuming animal protein below the quantity required by them. JARD Vol.

  1. The Tomato Terpene Synthase Gene Family1[W][OA

    Science.gov (United States)

    Falara, Vasiliki; Akhtar, Tariq A.; Nguyen, Thuong T.H.; Spyropoulou, Eleni A.; Bleeker, Petra M.; Schauvinhold, Ines; Matsuba, Yuki; Bonini, Megan E.; Schilmiller, Anthony L.; Last, Robert L.; Schuurink, Robert C.; Pichersky, Eran

    2011-01-01

    Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far. PMID:21813655

  2. Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila.

    Directory of Open Access Journals (Sweden)

    Praveen Balabaskaran Nina

    2010-07-01

    Full Text Available The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1 sector catalyzes ATP synthesis, whereas the F(o sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1 and F(o sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a

  3. Sucrose Phosphate Synthase and Sucrose Accumulation at Low Temperature 1

    Science.gov (United States)

    Guy, Charles L.; Huber, Joan L. A.; Huber, Steven C.

    1992-01-01

    The influence of growth temperature on the free sugar and sucrose phosphate synthase content and activity of spinach (Spinacia oleracea) leaf tissue was studied. When plants were grown at 25°C for 3 weeks and then transferred to a constant 5°C, sucrose, glucose, and fructose accumulated to high levels during a 14-d period. Predawn sugar levels increased from 14- to 20-fold over the levels present at the outset of the low-temperature treatment. Sucrose was the most abundant free sugar before, during, and after exposure to 5°C. Leaf sucrose phosphate synthase activity was significantly increased by the low-temperature treatment, whereas sucrose synthase and invertases were not. Synthesis of the sucrose phosphate synthase subunit was increased during and after low-temperature exposure and paralleled an increase in the steady-state level of the subunit. The increases in sucrose and its primary biosynthetic enzyme, sucrose phosphate synthase, are discussed in relation to adjustment of metabolism to low nonfreezing temperature and freezing stress tolerance. Images Figure 1 Figure 2 Figure 3 PMID:16652990

  4. Multi-level learning: improving the prediction of protein, domain and residue interactions by allowing information flow between levels

    Directory of Open Access Journals (Sweden)

    McDermott Drew

    2009-08-01

    Full Text Available Abstract Background Proteins interact through specific binding interfaces that contain many residues in domains. Protein interactions thus occur on three different levels of a concept hierarchy: whole-proteins, domains, and residues. Each level offers a distinct and complementary set of features for computationally predicting interactions, including functional genomic features of whole proteins, evolutionary features of domain families and physical-chemical features of individual residues. The predictions at each level could benefit from using the features at all three levels. However, it is not trivial as the features are provided at different granularity. Results To link up the predictions at the three levels, we propose a multi-level machine-learning framework that allows for explicit information flow between the levels. We demonstrate, using representative yeast interaction networks, that our algorithm is able to utilize complementary feature sets to make more accurate predictions at the three levels than when the three problems are approached independently. To facilitate application of our multi-level learning framework, we discuss three key aspects of multi-level learning and the corresponding design choices that we have made in the implementation of a concrete learning algorithm. 1 Architecture of information flow: we show the greater flexibility of bidirectional flow over independent levels and unidirectional flow; 2 Coupling mechanism of the different levels: We show how this can be accomplished via augmenting the training sets at each level, and discuss the prevention of error propagation between different levels by means of soft coupling; 3 Sparseness of data: We show that the multi-level framework compounds data sparsity issues, and discuss how this can be dealt with by building local models in information-rich parts of the data. Our proof-of-concept learning algorithm demonstrates the advantage of combining levels, and opens up

  5. Differential proteomics study of platelets in asymptomatic constitutional macrothrombocytopenia: altered levels of cytoskeletal proteins.

    Science.gov (United States)

    Karmakar, Shilpita; Saha, Sutapa; Banerjee, Debasis; Chakrabarti, Abhijit

    2015-01-01

    Harris platelet syndrome (HPS), also known as asymptomatic constitutional macrothrombocytopenia (ACMT), is an autosomal dominant platelet disorder characterized by mild-to-severe thrombocytopenia and giant platelets with normal platelet aggregation and absence of bleeding symptoms. We have attempted a comparative proteomics study for profiling of platelet proteins in healthy vs. pathological states to discover characteristic protein expression changes in macrothrombocytes and decipher the factors responsible for the functionally active yet morphologically distinct platelets. We have used 2-D gel-based protein separation techniques coupled with MALDI-ToF/ToF-based mass spectrometric identification and characterization of the proteins to investigate the differential proteome profiling of platelet proteins isolated from the peripheral blood samples of patients and normal volunteers. Our study revealed altered levels of actin-binding proteins such as myosin light chain, coactosin-like protein, actin-related protein 2/3 complex, and transgelin2 that hint toward the cytoskeletal changes necessary to maintain the structural and functional integrity of macrothrombocytes. We have also observed over expressed levels of peroxiredoxin2 that signifies the prevailing oxidative stress in these cells. Additionally, altered levels of protein disulfide isomerase and transthyretin provide insights into the measures adapted by the macrothrombocytes to maintain their normal functional activity. This first proteomics study of platelets from ACMT may provide an understanding of the structural stability and normal functioning of these platelets in spite of their large size. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

    Directory of Open Access Journals (Sweden)

    Kawamukai Makoto

    2004-11-01

    Full Text Available Abstract Background Isopentenyl diphosphate (IPP, a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

  7. Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics*

    Science.gov (United States)

    Zhang, Xi

    2015-01-01

    The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea–trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting—not destroying—structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. PMID:26081834

  8. Impact of High-Level Expression of Heterologous Protein on Lactococcus lactis Host.

    Science.gov (United States)

    Kim, Mina; Jin, Yerin; An, Hyun-Joo; Kim, Jaehan

    2017-07-28

    The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N -acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.

  9. Polyketide synthase from Fusarium

    DEFF Research Database (Denmark)

    Kvesel, Kasper; Wimmer, Reinhard; Sørensen, Jens Laurids

    described, even fewer from fungi and none from Fusarium species. Multidomain proteins can be quite challenging to work with, which is why the project intends to solve the 3D-structures of single domains of PKS’s. In this project, the plan is to clone, express and purify the Acyl-carrier protein (ACP) domain...... from PKS6 in Fusarium graminearum for structural analysis....

  10. Genetic regulation ofmethylation and IL1RL1-a protein levels in asthma

    NARCIS (Netherlands)

    Dijk, F Nicole; Xu, Chengjian; Melén, Erik; Carsin, Anne-Elie; Kumar, Asish; Nolte, Ilja M; Gruzieva, Olena; Pershagen, Goran; Grotenboer, Neomi S; Savenije, Olga E M; Antó, Josep Maria; Lavi, Iris; Dobaño, Carlota; Bousquet, Jean; van der Vlies, Pieter; van der Valk, Ralf J P; de Jongste, Johan C; Nawijn, Martijn C; Guerra, Stefano; Postma, Dirkje S; Koppelman, Gerard H

    2018-01-01

    Interleukin-1 receptor-like 1 (IL1RL1) is an important asthma gene. (Epi)genetic regulation ofIL1RL1protein expression has not been established. We assessed the association betweenIL1RL1single nucleotide polymorphisms (SNPs),IL1RL1methylation and serum IL1RL1-a protein levels, and aimed to identify

  11. Effect of different protein levels on the growth performance of African ...

    African Journals Online (AJOL)

    There were no significant differences (P.0.05) among treatments in initial body weight, protein efficiency ratio, average shell weight, average visceral weight, average edible weight and feed cost per kg weight gain. The results obtained in this study show that dietary protein level of about 22% is adequate for the growth of ...

  12. Class II recombinant phosphoribosyl diphosphate synthase from spinach

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows...... an unusual low specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and UTP in addition to ATP. The kinetic mechanism of the enzyme is an ordered steady state Bi Bi mechanism with K(ATP) and K(Rib-5-P) values of 170 and 110 micrometer, respectively, and a V(max) value of 13.1 micromol (min x...... mg of protein)(-1). The enzyme has an absolute requirement for magnesium ions, and maximal activity is obtained at 40 degrees C at pH 7.6....

  13. Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment

    International Nuclear Information System (INIS)

    Tanaka, Hiroaki; Tsurumura, Toshiharu; Aritake, Kosuke; Furubayashi, Naoki; Takahashi, Sachiko; Yamanaka, Mari; Hirota, Erika; Sano, Satoshi; Sato, Masaru; Kobayashi, Tomoyuki; Tanaka, Tetsuo; Inaka, Koji; Urade, Yoshihiro

    2011-01-01

    Crystals of hematopoietic prostaglandin D synthase grown in microgravity show improved quality. Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein

  14. Bioinformatics analysis of the phytoene synthase gene in cabbage (Brassica oleracea var. capitata)

    Science.gov (United States)

    Sun, Bo; Jiang, Min; Xue, Shengling; Zheng, Aihong; Zhang, Fen; Tang, Haoru

    2018-04-01

    Phytoene Synthase (PSY) is an important enzyme in carotenoid biosynthesis. Here, the Brassica oleracea var. capitata PSY (BocPSY) gene sequences were obtained from Brassica database (BRAD), and preformed for bioinformatics analysis. The BocPSY1, BocPSY2 and BocPSY3 genes mapped to chromosomes 2,3 and 9, and contains an open reading frame of 1,248 bp, 1,266 bp and 1,275 bp that encodes a 415, 421, 424 amino acid protein, respectively. Subcellular localization predicted all BocPSY genes were in the chloroplast. The conserved domain of the BocPSY protein is PLN02632. Homology analysis indicates that the levels of identity among BocPSYs were all more than 85%, and the PSY protein is apparently conserved during plant evolution. The findings of the present study provide a molecular basis for the elucidation of PSY gene function in cabbage.

  15. High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes.

    Directory of Open Access Journals (Sweden)

    Mariela V Catone

    Full Text Available Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB, a short chain length polyhydroxyalkanoate (sclPHA infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA. All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC in comparison with the mclPHA core genome genes (phaC1 and phaC2 indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases.

  16. Modulation of GDP-fucose level for generating proteins with reduced rate of fucosylation (WO2010141855).

    Science.gov (United States)

    Taupin, Philippe

    2011-09-01

    The application (WO2010141855) is in the field of glycobiology, and involves the control of the rate of fucosylation of proteins by exogenous factors. It aims at controlling the rate of protein fucosylation with inhibitors (drugs or nucleic acid antagonists) of enzymes involved in the synthesis of GDP-fucose. Mammalian cell lines were cultured in the presence of inhibitors, for example, siRNA. The rates of GDP-fucose in cells and during protein fucosylation were characterized. The level of protein fucosylation decreases rapidly in response to a decrease in GDP-fucose level. The relationship between the rate of fucosylation of proteins and the level of GDP-fucose in a cell is non-linear. Reduction in the rate of protein fucosylation can be achieved with a minimal reduction of the level of GDP-fucose in cells. The paradigm may be used to synthesize proteins and antibodies, with a reduced rate of fucosylation. The application claims that the use of drugs or nucleic acid antagonists that inhibit the enzymes involved in GDP-fucose biosynthesis optimizes the level of GDP-fucose present in cells, and reduces the rate of fucosylation of glycoproteins.

  17. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  18. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.

    Science.gov (United States)

    Bechard, Matthew E.; Chhatwal, Sonya; Garcia, Rosemarie E.; Rasche, Madeline E.

    2003-01-01

    Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  19. Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA–Protein Interactions at the Single-Molecule Level

    Directory of Open Access Journals (Sweden)

    Huijun Xue

    2017-10-01

    Full Text Available Much of our knowledge in conventional biochemistry has derived from bulk assays. However, many stochastic processes and transient intermediates are hidden when averaged over the ensemble. The powerful technique of single-molecule fluorescence microscopy has made great contributions to the understanding of life processes that are inaccessible when using traditional approaches. In single-molecule studies, quantum dots (Qdots have several unique advantages over other fluorescent probes, such as high brightness, extremely high photostability, and large Stokes shift, thus allowing long-time observation and improved signal-to-noise ratios. So far, however, there is no convenient way to label proteins purified from budding yeast with Qdots. Based on BirA–Avi and biotin–streptavidin systems, we have established a simple method to acquire a Qdot-labeled protein and visualize its interaction with DNA using total internal reflection fluorescence microscopy. For proof-of-concept, we chose replication protein A (RPA and origin recognition complex (ORC as the proteins of interest. Proteins were purified from budding yeast with high biotinylation efficiency and rapidly labeled with streptavidin-coated Qdots. Interactions between proteins and DNA were observed successfully at the single-molecule level.

  20. Nitrogen balance study in young Nigerian adult males using four levels of protein intake.

    Science.gov (United States)

    Atinmo, T; Mbofung, C M; Egun, G; Osotimehin, B

    1988-11-01

    1. The present study was carried out to estimate precisely, via the nitrogen balance technique, the protein requirement of Nigerians (earlier estimated via the obligatory N method) using graded levels of protein intake. 2. Fifteen medical students of the University of Ibadan who volunteered to participate in the study were given graded levels of protein (0.3, 0.45, 0.6 and 0.75 g/kg body-weight per d) derived from foods similar to those usually consumed by the subjects. 3. Each subject was given each of the dietary protein levels for a period of 10 d. Subjects were divided into two groups and the feeding pattern followed a criss-cross design with one group starting with the highest level of protein intake (0.3 g). Mean energy intake during each of the eleven experimental periods was maintained at 0.2 MJ/kg per d. After an initial 5 d adaptation period in each experimental period, 24 h urine and faecal samples were collected in marked containers for five consecutive days for N determination. 4. Mean N balance during consumption of the four protein levels (0.30, 0.45, 0.6 and 0.75 g/kg) were -11.02 (SD 8.07), -9.90 (SD 6.64), +9.70 (SD 4.15) and +5.13 (SD 4.62) respectively. Using regression analysis, the mean daily N requirement was estimated at 110.25 mg N/kg body-weight (0.69 g protein/kg body-weight). Estimates of allowances for individual variations to cover 97.5% of the population adjusted this value to 0.75 g protein/kg body-weight. Net protein utilization for the diet at maintenance level was estimated at 57.5.

  1. Identifying the catalytic components of cellulose synthase and the maize mixed-linkage beta-glucan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Nicholas C Carpita

    2009-04-20

    Five specific objectives of this project are to develop strategies to identify the genes that encode the catalytic components of "mixed-linkage" (1→3),(1→4)-beta-D-glucans in grasses, to determine the protein components of the synthase complex, and determine the biochemical mechanism of synthesis. We have used proteomic approaches to define intrinsic and extrinsic polypeptides of Golgi membranes that are associated with polysaccharide synthesis and trafficking. We were successful in producing recombinant catalytic domains of cellulose synthase genes and discovered that they dimerize upon concentration, indicating that two CesA proteins form the catalytic unit. We characterized a brittle stalk2 mutant as a defect in a COBRA-like protein that results in compromised lignin-cellulose interactions that decrease tissue flexibility. We used virus-induced gene silencing of barley cell wall polysaccharide synthesis by BSMV in an attempt to silence specific members of the cellulose synthase-like gene family. However, we unexpectedly found that regardless of the specificity of the target gene, whole gene interaction networks were silenced. We discovered the cause to be an antisense transcript of the cellulose synthase gene initiated small interfering RNAs that spread silencing to related genes.

  2. Virtual Screening of Novel Glucosamine-6-Phosphate Synthase Inhibitors.

    Science.gov (United States)

    Lather, Amit; Sharma, Sunil; Khatkar, Anurag

    2018-01-01

    affinities and interaction between the inhibitors and the target proteins (G-6-P synthase) by using Glide software (Schrodinger Inc. U.S.A.-Maestro version 10.2). Grid-based Ligand Docking with Energetic (Glide) is one of the most accurate docking softwares available for ligand-protein, protein-protein binding studies. A library of hundreds of available ligands was docked against targeted proteins G-6-P synthase having PDB ID 1moq. Results of docking are shown in Table 1 and Table 2. Results of G-6-P synthase docking showed that some compounds were found to have comparable docking score and binding energy (kj/mol) as compared to standard antibiotics. Many of the ligands showed hydrogen bond interaction, hydrophobic interactions, electrostatic interactions, ionic interactions and π- π stacking with the various amino acid residues in the binding pockets of G-6-P synthase. The docking study estimated free energy of binding, binding pose andglide score and all these parameters provide a promising tool for the discovery of new potent natural inhibitors of G-6-P synthase. These G-6-P synthase inhibitors could further be used as antimicrobials. Here, a detailed binding analysis and new insights of inhibitors from various classes of molecules were docked in binding cavity of G-6-P synthase. ADME and toxicity prediction of these compounds will further accentuate us to study these compounds in vivo. This information will possibly present further expansion of effective antimicrobials against several microbial infections. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Perinatal exposure to BDE-99 causes decreased protein levels of cyclin D1 via GSK3β activation and increased ROS production in rat pup livers.

    Science.gov (United States)

    Blanco, Jordi; Mulero, Miquel; Domingo, Jose L; Sanchez, Domènec J

    2014-02-01

    We here examined the potential liver toxicity in rat pups from dams exposed during the gestational and lactation periods to 2,2',4,4',5-pentabromodiphenyl ether (BDE-99). Dams were exposed to 0, 1, and 2mg/kg/day of BDE-99 from gestation day 6 to postnatal day 21. When the pups were weaning, the liver from 1 pup of each litter was excised to evaluate oxidative stress markers and the messenger RNA (mRNA) expression of multiple cytochrome P450 (CYP) isoforms. To determine whether thyroid hormone (TH) was disrupted, the protein and mRNA expressions of several TH receptor (TR) isoforms, as well as the protein levels of cyclin D1 and the phosphorylated protein kinases Akt and glycogen synthase kinase 3 beta (GSK3β), were evaluated. Perinatal exposure to BDE-99 produced decreased levels of cyclin D1 in rat pup livers. A decrease in the active form of Akt and an increase in the active form of GSK3β were observed. The decreased Akt pathway may be due to a potential disruption of the nongenomic actions of TH by BDE-99 and its metabolites. This possible TH disruption was noted as a decrease in TR isoforms expression. By contrast, we observed an upregulation of CYP2B1 gene expression, which is correlated with an increase in reactive oxygen species production. This outcome indicates activation of the nuclear constitutive androstane receptor, which could induce the expression of other enzymes capable of metabolizing TH. The present findings support the hypothesis that perinatal exposure to PBDEs, at levels found in humans, may have serious implications for metabolic processes in rat pup livers.

  4. Intestinal nitric oxide synthase activity changes during experimental colon obstruction.

    Science.gov (United States)

    Palásthy, Zsolt; Kaszaki, József; Lázár, György; Nagy, Sándor; Boros, Mihály

    2006-08-01

    The experiments in this study were designed to follow the time course of nitric oxide (NO) synthesis in the large bowel during acute mechanical ileus. Occlusion of the mid-transverse colon was maintained for 420 min in anesthetized dogs. Strain-gauge transducers were used to analyze motility changes on the hepatic and lienal flexures, respectively. Constitutive NO synthase (cNOS) and inducible NOS (iNOS) activities were determined in tissue biopsies, and plasma nitrite/nitrate (NOx) level was measured in the portal blood. Following completion of the baseline studies, the animals were treated with either 7-nitroindazole (7-NI, selective neuronal NOS inhibitor), or N-nitro-L-arginine (NNA, non-selective NOS inhibitor). In the sham-operated group the cNOS activities differed significantly in the oral and aboral tissue samples (oral: 102.9; versus aboral: 62.1 fmol/mg protein/min). The obstruction elicited a significant increase in portal NOx and elevated tissue inducible NO synthase (iNOS) activity. NNA treatment decreased the motility index in both intestinal segments for 60 min, but 120 min later the motility index was significantly elevated (2.5-fold increase in the oral part, and 1.8-fold enhancement in the aboral segment, respectively). Treatment with 7-NI decreased the cNOS activity in the oral and aboral parts by approximately 40% and 70%, respectively, and suppressed the motility increase in the aboral colon segment. The motility of the colon was either significantly increased or decreased, depending on the type and selectivity of the NOS inhibitor compounds applied. NO of neuronal origin is a transmitter that stimulates peristaltic activity; but an increased iNOS/nNOS ratio significantly moderates the obstruction-induced motility increase.

  5. Preparation, crystallization and preliminary X-ray analysis of the methionine synthase (MetE) from Streptococcus mutans

    International Nuclear Information System (INIS)

    Fu, Tian-Min; Zhang, Xiao-Yan; Li, Lan-Fen; Liang, Yu-He; Su, Xiao-Dong

    2006-01-01

    Methionine synthase (MetE) from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.2 Å resolution. The Streptococcus mutans metE gene encodes methionine synthase (MetE), which catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to homocysteine in the last step of methionine synthesis. metE was cloned into pET28a and the gene product was expressed at high levels in the Escherichia coli strain BL21 (DE3). MetE was purified to homogeneity using Ni 2+ -chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.2 Å resolution. The crystal belongs to space group P2 1 , with unit-cell parameters a = 52.85, b = 99.48, c = 77.88 Å, β = 94.55°

  6. Preparation, crystallization and preliminary X-ray analysis of the methionine synthase (MetE) from Streptococcus mutans

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Tian-Min; Zhang, Xiao-Yan; Li, Lan-Fen; Liang, Yu-He, E-mail: liangyh@pku.edu.cn; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871 (China); Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing 100871 (China)

    2006-10-01

    Methionine synthase (MetE) from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.2 Å resolution. The Streptococcus mutans metE gene encodes methionine synthase (MetE), which catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to homocysteine in the last step of methionine synthesis. metE was cloned into pET28a and the gene product was expressed at high levels in the Escherichia coli strain BL21 (DE3). MetE was purified to homogeneity using Ni{sup 2+}-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.2 Å resolution. The crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 52.85, b = 99.48, c = 77.88 Å, β = 94.55°.

  7. Protein intake and stress levels in nurses and housewives of Pakistan

    Science.gov (United States)

    Wattoo, Feroza Hamid; Memon, Muhammad Saleh; Memon, Allah Nawaz; Wattoo, Muhammad Hamid Sarwar; Asad, Muhammad Javaid; Siddique, Farzana

    2011-01-01

    Stress has many biological effects on human daily life. In the present study, dietary protein intake was correlated with the investigated stress levels of nurses and housewives of the targeted urban population. Age group ranged from 30 to 45 years and both the groups belonged to middle socioeconomic status. After calculations of environmental, psychological and physiological stresses, it was observed that the levels of stress in housewives were significantly higher than those of nurses. Recommended dietary allowances, RDA and actual protein intakes, API were also compared in both the groups. The found protein intake was less in housewives as compared to that of nurses. PMID:23961140

  8. Protein level affects the relative lysine requirement of growing rainbow trout (Oncorhynchus mykiss) fry.

    Science.gov (United States)

    Bodin, Noelie; Govaerts, Bernadette; Abboudi, Tarik; Detavernier, Christel; De Saeger, Sarah; Larondelle, Yvan; Rollin, Xavier

    2009-07-01

    The effect of two digestible protein levels (310 and 469 g/kg DM) on the relative lysine (Lys; g Lys/kg DM or g Lys/100 g protein) and the absolute Lys (g Lys intake/kg 0.75 per d) requirements was studied in rainbow trout fry using a dose-response trial. At each protein level, sixteen isoenergetic (22-23 MJ digestible energy/kg DM) diets were tested, involving a full range (2-70 g/kg DM) of sixteen Lys levels. Each diet was given to one group of sixty rainbow trout fry (mean initial body weight 0.78 g) reared at 15 degrees C for 31 feeding d. The Lys requirements were estimated based on the relationships between weight, protein, and Lys gains (g/kg 0.75 per d) and Lys concentration (g/kg DM or g/100 g protein) or Lys intake (g/kg 0.75 per d), using the broken-line model (BLM) and the non-linear four-parameter saturation kinetics model (SKM-4). Both the model and the response criterion chosen markedly impacted the relative Lys requirement. The relative Lys requirement for Lys gain of rainbow trout estimated with the BLM (and SKM-4 at 90 % of the maximum response) increased from 16.8 (19.6) g/kg DM at a low protein level to 23.4 (24.5) g/kg DM at a high protein level. However, the dietary protein content affected neither the absolute Lys requirement nor the relative Lys requirement expressed as g Lys/100 g protein nor the Lys requirement for maintenance (21 mg Lys/kg 0.75 per d).

  9. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

    Science.gov (United States)

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W; Grubert, Fabian; Candille, Sophie I; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L; Tang, Hua; Ricci, Emiliano; Snyder, Michael P

    2015-11-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. © 2015 Cenik et al.; Published by Cold Spring Harbor Laboratory Press.

  10. Heightened systemic levels of neutrophil and eosinophil granular proteins in pulmonary tuberculosis and reversal following treatment.

    Science.gov (United States)

    Moideen, Kadar; Kumar, Nathella Pavan; Nair, Dina; Banurekha, Vaithilingam V; Bethunaickan, Ramalingam; Babu, Subash

    2018-04-09

    Granulocytes are activated during tuberculosis (TB) infection and act as immune effector cells and granulocyte responses are implicated in TB pathogenesis. Plasma levels of neutrophil and eosinophil granular proteins provide an indirect measure of degranulation. In this study, we wanted to examine the levels of neutrophil and eosinophil granular proteins in individuals with pulmonary tuberculosis (PTB) and to compare them with the levels in latent TB (LTB) individuals. Hence, we measured the plasma levels of myeloperoxidase (MPO), neutrophil elastase, and proteinase-3; major basic protein (MBP), eosinophil derived neurotoxin (EDN), eosinophil cationic protein (ECP) and eosinophil peroxidase (EPX) in these individuals. Finally, we also measured the levels of all of these parameters in PTB individuals following anti-tuberculosis (ATT) treatment. Our data reveal that PTB individuals are characterized by significantly higher plasma levels of MPO, elastase, human proteinase 3 as well as MBP and EDN in comparison to LTB individuals. Our data also reveal that ATT resulted in reversal of all of these changes, indicating an association with TB disease. Finally, our data show that the systemic levels of MPO and proteinase-3 can significantly discriminate PTB from LTB individuals. Thus, our data suggest that neutrophil and eosinophil granular proteins could play a potential role in the innate immune response and therefore, the pathogenesis of pulmonary TB. Copyright © 2018 American Society for Microbiology.

  11. Changes in human parotid salivary protein and sialic acid levels during pregnancy.

    Science.gov (United States)

    D'Alessandro, S; Curbelo, H M; Tumilasci, O R; Tessler, J A; Houssay, A B

    1989-01-01

    Saliva was collected with a Carlson-Crittenden device, under citric acid stimulation, in 107 pregnant women, 9 puerperal and 7 non-pregnant controls. No significant changes were found in salivary flow rate, pH and amylase levels. The total protein levels were decreased during pregnancy and the puerperium. The sialic acid levels decreased gradually but markedly during pregnancy, returning to normal levels in the puerperium. These changes in parotid saliva may be related to the hormonal changes of pregnancy.

  12. Fasting Lipoprotein Lipase Protein Levels Can Predict a Postmeal Increment of Triglyceride Levels in Fasting Normohypertriglyceridemic Subjects.

    Science.gov (United States)

    Tsuzaki, Kokoro; Kotani, Kazuhiko; Yamada, Kazunori; Sakane, Naoki

    2016-09-01

    Although a postprandial increment in triglyceride (TG) levels is considered to be a risk factor for atherogenesis, tests (e.g., fat load) to assess postprandial changes in TG levels cannot be easily applied to clinical practice. Therefore, fasting markers that predict postprandial TG states are needed to be developed. One current candidate is lipoprotein lipase (LPL) protein, a molecule that hydrides TGs. This study investigated whether fasting LPL levels could predict postprandial TG levels. A total of 17 subjects (11 men, 6 women, mean age 52 ± 11 years) with normotriglyceridemia during fasting underwent the meal test. Several fasting parameters, including LPL, were measured for the area under the curve of postprandial TGs (AUC-TG). The subjects' mean fasting TG level was 1.30 mmol/l, and their mean LPL level was 41.6 ng/ml. The subjects' TG levels increased after loading (they peaked after two postprandial hours). Stepwise multiple regression analysis demonstrated that fasting TG levels were a predictor of the AUC-TG. In addition, fasting LPL mass levels were found to be a predictor of the AUC-TG (β = 0.65, P fasting TG levels. Fasting LPL levels may be useful to predict postprandial TG increment in this population. © 2015 Wiley Periodicals, Inc.

  13. Characterization of the human gene (TBXAS1) encoding thromboxane synthase.

    Science.gov (United States)

    Miyata, A; Yokoyama, C; Ihara, H; Bandoh, S; Takeda, O; Takahashi, E; Tanabe, T

    1994-09-01

    The gene encoding human thromboxane synthase (TBXAS1) was isolated from a human EMBL3 genomic library using human platelet thromboxane synthase cDNA as a probe. Nucleotide sequencing revealed that the human thromboxane synthase gene spans more than 75 kb and consists of 13 exons and 12 introns, of which the splice donor and acceptor sites conform to the GT/AG rule. The exon-intron boundaries of the thromboxane synthase gene were similar to those of the human cytochrome P450 nifedipine oxidase gene (CYP3A4) except for introns 9 and 10, although the primary sequences of these enzymes exhibited 35.8% identity each other. The 1.2-kb of the 5'-flanking region sequence contained potential binding sites for several transcription factors (AP-1, AP-2, GATA-1, CCAAT box, xenobiotic-response element, PEA-3, LF-A1, myb, basic transcription element and cAMP-response element). Primer-extension analysis indicated the multiple transcription-start sites, and the major start site was identified as an adenine residue located 142 bases upstream of the translation-initiation site. However, neither a typical TATA box nor a typical CAAT box is found within the 100-b upstream of the translation-initiation site. Southern-blot analysis revealed the presence of one copy of the thromboxane synthase gene per haploid genome. Furthermore, a fluorescence in situ hybridization study revealed that the human gene for thromboxane synthase is localized to band q33-q34 of the long arm of chromosome 7. A tissue-distribution study demonstrated that thromboxane synthase mRNA is widely expressed in human tissues and is particularly abundant in peripheral blood leukocyte, spleen, lung and liver. The low but significant levels of mRNA were observed in kidney, placenta and thymus.

  14. Performance of juvenile mojarra supplied with feed containing varying levels of crude protein

    Directory of Open Access Journals (Sweden)

    Ricardo Henrique Bastos de Souza

    2016-04-01

    Full Text Available ABSTRACT The growth of the Brazilian aquaculture has stimulated the development of the productive chain of native species, including marine environment. The objective of this study was to evaluate the growth performance of juvenile mojarra fish (Diapterus rhombeus fed diets containing different concentrations of crude protein (32, 36, 40 and 44 g 100 g-1. The 80 juvenile mojarra (7.2±1.5 g were kept in 16 circular tanks (150 L. The study design used was completely randomized with four treatments and four repetitions. The fish were fed four times a day. At the end of the experiment (60 days the final weight, feed intake, weight gain (WG, feed:gain ratio (FGR, protein efficiency rate (PER, energy efficiency rate, specific growth, survival rate and, body composition were evaluated. It was verified significant effect of protein level on the WG, with the best value at the level of 38.20 g 100 g-1 of crude protein. For FGR, the best estimated value occurred with 38.06 g 100 g-1 of crude protein, similar to that reported for the PER (38.91 g 100 g-1. The other performance parameters and body composition were not influenced by crude protein levels. Diet crude protein concentrations between 38.06 and 38.91 g 100 g-1 provide the best performance indices for juvenile mojarra.

  15. Exploring sequence characteristics related to high-level production of secreted proteins in Aspergillus niger.

    Directory of Open Access Journals (Sweden)

    Bastiaan A van den Berg

    Full Text Available Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large set, over 600 homologous and nearly 2,000 heterologous fungal genes, were overexpressed in Aspergillus niger using a standardized expression cassette and scored for high versus no production. Subsequently, sequence-based machine learning techniques were applied for identifying relevant DNA and protein sequence features. The amino-acid composition of the protein sequence was found to be most predictive and interpretation revealed that, for both homologous and heterologous gene expression, the same features are important: tyrosine and asparagine composition was found to have a positive correlation with high-level production, whereas for unsuccessful production, contributions were found for methionine and lysine composition. The predictor is available online at http://bioinformatics.tudelft.nl/hipsec. Subsequent work aims at validating these findings by protein engineering as a method for increasing expression levels per gene copy.

  16. Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.

    Science.gov (United States)

    Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T

    2017-01-01

    A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.

  17. Functional Characterization of Sesquiterpene Synthase from Polygonum minus

    Directory of Open Access Journals (Sweden)

    Su-Fang Ee

    2014-01-01

    Full Text Available Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS has a complete open reading frame (ORF of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of β-sesquiphellandrene.

  18. Alcohol-Binding Sites in Distinct Brain Proteins: The Quest for Atomic Level Resolution

    Science.gov (United States)

    Howard, Rebecca J.; Slesinger, Paul A.; Davies, Daryl L.; Das, Joydip; Trudell, James R.; Harris, R. Adron

    2011-01-01

    Defining the sites of action of ethanol on brain proteins is a major prerequisite to understanding the molecular pharmacology of this drug. The main barrier to reaching an atomic-level understanding of alcohol action is the low potency of alcohols, ethanol in particular, which is a reflection of transient, low-affinity interactions with their targets. These mechanisms are difficult or impossible to study with traditional techniques such as radioligand binding or spectroscopy. However, there has been considerable recent progress in combining X-ray crystallography, structural modeling, and site-directed mutagenesis to define the sites and mechanisms of action of ethanol and related alcohols on key brain proteins. We review such insights for several diverse classes of proteins including inwardly rectifying potassium, transient receptor potential, and neurotransmit-ter-gated ion channels, as well as protein kinase C epsilon. Some common themes are beginning to emerge from these proteins, including hydrogen bonding of the hydroxyl group and van der Waals interactions of the methylene groups of ethanol with specific amino acid residues. The resulting binding energy is proposed to facilitate or stabilize low-energy state transitions in the bound proteins, allowing ethanol to act as a “molecular lubricant” for protein function. We discuss evidence for characteristic, discrete alcohol-binding sites on protein targets, as well as evidence that binding to some proteins is better characterized by an interaction region that can accommodate multiple molecules of ethanol. PMID:21676006

  19. 2-Oxoglutarate levels control adenosine nucleotide binding by Herbaspirillum seropedicae PII proteins.

    Science.gov (United States)

    Oliveira, Marco A S; Gerhardt, Edileusa C M; Huergo, Luciano F; Souza, Emanuel M; Pedrosa, Fábio O; Chubatsu, Leda S

    2015-12-01

    Nitrogen metabolism in Proteobacteria is controlled by the Ntr system, in which PII proteins play a pivotal role, controlling the activity of target proteins in response to the metabolic state of the cell. Characterization of the binding of molecular effectors to these proteins can provide information about their regulation. Here, the binding of ATP, ADP and 2-oxoglutarate (2-OG) to the Herbaspirillum seropedicae PII proteins, GlnB and GlnK, was characterized using isothermal titration calorimetry. Results show that these proteins can bind three molecules of ATP, ADP and 2-OG with homotropic negative cooperativity, and 2-OG binding stabilizes the binding of ATP. Results also show that the affinity of uridylylated forms of GlnB and GlnK for nucleotides is significantly lower than that of the nonuridylylated proteins. Furthermore, fluctuations in the intracellular concentration of 2-OG in response to nitrogen availability are shown. Results suggest that under nitrogen-limiting conditions, PII proteins tend to bind ATP and 2-OG. By contrast, after an ammonium shock, a decrease in the 2-OG concentration is observed causing a decrease in the affinity of PII proteins for ATP. This phenomenon may facilitate the exchange of ATP for ADP on the ligand-binding pocket of PII proteins, thus it is likely that under low ammonium, low 2-OG levels would favor the ADP-bound state. © 2015 FEBS.

  20. Multi-level machine learning prediction of protein–protein interactions in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Julian Zubek

    2015-07-01

    Full Text Available Accurate identification of protein–protein interactions (PPI is the key step in understanding proteins’ biological functions, which are typically context-dependent. Many existing PPI predictors rely on aggregated features from protein sequences, however only a few methods exploit local information about specific residue contacts. In this work we present a two-stage machine learning approach for prediction of protein–protein interactions. We start with the carefully filtered data on protein complexes available for Saccharomyces cerevisiae in the Protein Data Bank (PDB database. First, we build linear descriptions of interacting and non-interacting sequence segment pairs based on their inter-residue distances. Secondly, we train machine learning classifiers to predict binary segment interactions for any two short sequence fragments. The final prediction of the protein–protein interaction is done using the 2D matrix representation of all-against-all possible interacting sequence segments of both analysed proteins. The level-I predictor achieves 0.88 AUC for micro-scale, i.e., residue-level prediction. The level-II predictor improves the results further by a more complex learning paradigm. We perform 30-fold macro-scale, i.e., protein-level cross-validation experiment. The level-II predictor using PSIPRED-predicted secondary structure reaches 0.70 precision, 0.68 recall, and 0.70 AUC, whereas other popular methods provide results below 0.6 threshold (recall, precision, AUC. Our results demonstrate that multi-scale sequence features aggregation procedure is able to improve the machine learning results by more than 10% as compared to other sequence representations. Prepared datasets and source code for our experimental pipeline are freely available for download from: http://zubekj.github.io/mlppi/ (open source Python implementation, OS independent.

  1. Level of C - reactive protein as an indicator for prognosis of premature uterine contractions.

    Science.gov (United States)

    Najat Nakishbandy, Bayar M; Barawi, Sabat A M

    2014-01-01

    high concentrations of maternal C-reactive protein have been associated with adverse pregnancy outcome, and premature uterine contraction may be predicted by elevated levels of C-reactive protein. This may ultimately be simple and cost-effective enough to introduce as a low-risk screening program. an observational case control study was performed from May 1st, 2010 to December 1st, 2010 at Maternity Teaching Hospital-Erbil/ Kurdistan Region/ Iraq. The sample size was (200) cases. Hundred of them were presented with premature uterine contractions at 24(+0)-36(+6) weeks. The other hundred were control group at same gestational ages. The level of C-reactive protein was determined in both groups and both groups were followed till delivery. (93) out of (100) women with premature uterine contractions had elevated level of C-Reactive protein and 91% delivered prematurely while in the control group only (9) out of (100) women had elevated level of C-reactive protein and only 8% of them delivered preterm. Differences were statistically highly significant. C-reactive protein can be used as a biomarker in prediction of premature delivery when it is associated with premature uterine contractions. As well it can be used as a screening test to detect cases that are at risk of premature delivery.

  2. Germacrene A Synthase in Yarrow (Achillea millefolium Is an Enzyme with Mixed Substrate Specificity: Gene Cloning, Functional Characterization and Expression Analysis

    Directory of Open Access Journals (Sweden)

    Leila ePazouki

    2015-03-01

    Full Text Available Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5 residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS. The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3, functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP, while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP. Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes.

  3. Harmful effect of protein difficiency on lipids, glucose, insulin and estradiol levels in female albino rats

    International Nuclear Information System (INIS)

    El-Mahdy, A.A.; El-Sherbiny, E.M.; Bayomi, M.M.

    2005-01-01

    The present study was undertaken to investigate the harmful effect of protein deficient diet on some biochemical activities in serum of female rats. Protein malnutrition is a well known socioeconomic problem in different parts of the world. Many studies were investigated on the biological parameters following protein malnutrition in human and experimental animals. Forty albino female rats were divided into 3 groups. The first group (10 rats) fed 18% protein diet and served as normal control and the other two groups, each contains 15 rats, fed 5% protein for 21 and 45 days, respectively, and served as malnourished groups. The results showed significant decrease in total body weight, serum glucose, insulin and estradiol levels in the third group as well as decrease in the total cholesterol, HDL-cholesterol, LDL-cholesterol and VLDL-cholesterol and triglycerides concentrations that compared to normal control rats

  4. Protein Consumption and the Elderly: What Is the Optimal Level of Intake?

    Directory of Open Access Journals (Sweden)

    Jamie I. Baum

    2016-06-01

    Full Text Available Maintaining independence, quality of life, and health is crucial for elderly adults. One of the major threats to living independently is the loss of muscle mass, strength, and function that progressively occurs with aging, known as sarcopenia. Several studies have identified protein (especially the essential amino acids as a key nutrient for muscle health in elderly adults. Elderly adults are less responsive to the anabolic stimulus of low doses of amino acid intake compared to younger individuals. However, this lack of responsiveness in elderly adults can be overcome with higher levels of protein (or essential amino acid consumption. The requirement for a larger dose of protein to generate responses in elderly adults similar to the responses in younger adults provides the support for a beneficial effect of increased protein in older populations. The purpose of this review is to present the current evidence related to dietary protein intake and muscle health in elderly adults.

  5. Predicting protein folding rate change upon point mutation using residue-level coevolutionary information.

    Science.gov (United States)

    Mallik, Saurav; Das, Smita; Kundu, Sudip

    2016-01-01

    Change in folding kinetics of globular proteins upon point mutation is crucial to a wide spectrum of biological research, such as protein misfolding, toxicity, and aggregations. Here we seek to address whether residue-level coevolutionary information of globular proteins can be informative to folding rate changes upon point mutations. Generating residue-level coevolutionary networks of globular proteins, we analyze three parameters: relative coevolution order (rCEO), network density (ND), and characteristic path length (CPL). A point mutation is considered to be equivalent to a node deletion of this network and respective percentage changes in rCEO, ND, CPL are found linearly correlated (0.84, 0.73, and -0.61, respectively) with experimental folding rate changes. The three parameters predict the folding rate change upon a point mutation with 0.031, 0.045, and 0.059 standard errors, respectively. © 2015 Wiley Periodicals, Inc.

  6. Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

    Science.gov (United States)

    Fischer, Jana; Kleinau, Gunnar; Rutz, Claudia; Zwanziger, Denise; Khajavi, Noushafarin; Müller, Anne; Rehders, Maren; Brix, Klaudia; Worth, Catherine L; Führer, Dagmar; Krude, Heiko; Wiesner, Burkhard; Schülein, Ralf; Biebermann, Heike

    2018-06-01

    G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G q/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

  7. Protein synthesis levels are increased in a subset of individuals with Fragile X syndrome

    DEFF Research Database (Denmark)

    Jacquemont, Sébastien; Pacini, Laura; Jønch, Aia E

    2018-01-01

    architecture and plasticity. Preclinical studies revealed that pharmacological interventions restore those deficits, which are thought to mediate the FXS cognitive and behavioral symptoms. Here we characterized the de novo rate of protein synthesis in patients with FXS and their relationship with clinical...... severity. We measured the rate of protein synthesis in fibroblasts derived from 32 individuals with FXS and from 17 controls as well as in fibroblasts and primary neurons of 27 Fmr1 KO mice and 20 controls. Here we show that levels of protein synthesis are increased in fibroblasts of individuals with FXS...... and Fmr1 KO mice. However, this cellular phenotype displays a broad distribution and a proportion of fragile X individuals and Fmr1 KO mice do not show increased levels of protein synthesis, having measures in the normal range. Because the same Fmr1 KO animal measures in fibroblasts predict those...

  8. Do infants with cow's milk protein allergy have inadequate levels of vitamin D?

    Science.gov (United States)

    Silva, Cristiane M; Silva, Silvia A da; Antunes, Margarida M de C; Silva, Gisélia Alves Pontes da; Sarinho, Emanuel Sávio Cavalcanti; Brandt, Katia G

    To verify whether infants with cow's milk protein allergy have inadequate vitamin D levels. This cross-sectional study included 120 children aged 2 years or younger, one group with cow's milk protein allergy and a control group. The children were recruited at the pediatric gastroenterology, allergology, and pediatric outpatient clinics of a university hospital in the Northeast of Brazil. A questionnaire was administered to the caregiver and blood samples were collected for vitamin D quantification. Vitamin D levels <30ng/mL were considered inadequate. Vitamin D level was expressed as mean and standard deviation, and the frequency of the degrees of sufficiency and other variables, as proportions. Infants with cow's milk protein allergy had lower mean vitamin D levels (30.93 vs.35.29ng/mL; p=0.041) and higher deficiency frequency (20.3% vs.8.2; p=0.049) than the healthy controls. Exclusively or predominantly breastfed infants with cow's milk protein allergy had higher frequency of inadequate vitamin D levels (p=0.002). Regardless of sun exposure time, the groups had similar frequencies of inadequate vitamin D levels (p=0.972). Lower vitamin D levels were found in infants with CMPA, especially those who were exclusively or predominantly breastfed, making these infants a possible risk group for vitamin D deficiency. Copyright © 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  9. Do infants with cow's milk protein allergy have inadequate levels of vitamin D?

    Directory of Open Access Journals (Sweden)

    Cristiane M. Silva

    Full Text Available Abstract Objective: To verify whether infants with cow's milk protein allergy have inadequate vitamin D levels. Methods: This cross-sectional study included 120 children aged 2 years or younger, one group with cow's milk protein allergy and a control group. The children were recruited at the pediatric gastroenterology, allergology, and pediatric outpatient clinics of a university hospital in the Northeast of Brazil. A questionnaire was administered to the caregiver and blood samples were collected for vitamin D quantification. Vitamin D levels <30 ng/mL were considered inadequate. Vitamin D level was expressed as mean and standard deviation, and the frequency of the degrees of sufficiency and other variables, as proportions. Results: Infants with cow's milk protein allergy had lower mean vitamin D levels (30.93 vs.35.29 ng/mL; p = 0.041 and higher deficiency frequency (20.3% vs.8.2; p = 0.049 than the healthy controls. Exclusively or predominantly breastfed infants with cow's milk protein allergy had higher frequency of inadequate vitamin D levels (p = 0.002. Regardless of sun exposure time, the groups had similar frequencies of inadequate vitamin D levels (p = 0.972. Conclusions: Lower vitamin D levels were found in infants with CMPA, especially those who were exclusively or predominantly breastfed, making these infants a possible risk group for vitamin D deficiency.

  10. Less is More: Membrane Protein Digestion Beyond Urea-Trypsin Solution for Next-level Proteomics.

    Science.gov (United States)

    Zhang, Xi

    2015-09-01

    The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea-trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting-not destroying-structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. BDNF downregulates 5-HT(2A) receptor protein levels in hippocampal cultures

    DEFF Research Database (Denmark)

    Trajkovska, V; Santini, M A; Marcussen, Anders Bue

    2009-01-01

    Both brain-derived neurotrophic factor (BDNF) and the serotonin receptor 2A (5-HT(2A)) have been related to depression pathology. Specific 5-HT(2A) receptor changes seen in BDNF conditional mutant mice suggest that BDNF regulates the 5-HT(2A) receptor level. Here we show a direct effect of BDNF...... on 5-HT(2A) receptor protein levels in primary hippocampal neuronal and mature hippocampal organotypic cultures exposed to different BDNF concentrations for either 1, 3, 5 or 7 days. In vivo effects of BDNF on hippocampal 5-HT(2A) receptor levels were further corroborated in (BDNF +/-) mice...... with reduced BDNF levels. In primary neuronal cultures, 7 days exposure to 25 and 50ng/mL BDNF resulted in downregulation of 5-HT(2A), but not of 5-HT(1A), receptor protein levels. The BDNF-associated downregulation of 5-HT(2A) receptor levels was also observed in mature hippocampal organotypic cultures...

  12. Investigation of the molecular level interactions between mucins and food proteins: Spectroscopic, tribological and rheological studies

    OpenAIRE

    Celebioglu, Hilal Yilmaz; Chronakis, Ioannis S.; Lee, Seunghwan; Guðjónsdóttir, María

    2017-01-01

    The thesis investigated the structure and molecular-level interaction of β-lactoglobulin (BLG) and mucins, representing major components of the dairy products and saliva/digestion systems, respectively. Mucins are long glycoprotein molecules responsible for the gel nature of the mucous layer covers epithelial surfaces throughout the body. A literature review of the interactions of different mucin types and saliva mucins with several food proteins and food protein emulsions, as well as their f...

  13. Urea metabolism in buffalo calves fed on rations containing two levels of crude protein

    International Nuclear Information System (INIS)

    Verma, D.N.; Singh, U.B.; Lal, M.; Varma, A.; Ranjhan, S.K.

    1974-01-01

    Urea entry rates into the body pools of Murrah Buffalo calves have been estimated using a single injection isotope dilution technique using 14 C-urea. The animals were fed two levels of crude proteins, namely, 13 percent lower and 19 percent higher than N.R.C. recommendations. Results show that the recycling of urea is significantly better in animals given low crude protein contents. (M.G.B.)

  14. Effects of dietary protein levels on length-weight relationships and ...

    African Journals Online (AJOL)

    Feeding trial involving different protein levels on length–weight relationships and condition factor of Clarias gariepinus was conducted in floating hapa system. Fingerlings (average weight, 4.50± 0.01g and average length, 8.0±0.2 cm) were randomly stocked at 20 fish/1m3. Five diets with crude protein: 40.0, 42.5, 45.0, 47.5 ...

  15. Interaction betwen Lead and Bone Protein to Affect Bone Calcium Level Using UV-Vis Spectroscopy

    Science.gov (United States)

    Noor, Z.; Azharuddin, A.; Aflanie, I.; Kania, N.; Suhartono, E.

    2018-05-01

    This present study aim to evaluate the interactions between lead (Pb) and with bone protein by UV-Vis approach. In addition, this prsent study also aim to investigate the effect of Pb on bone calcium (Ca) level. The present study was a true experimental study design to examine the impact of Pb exposure in bone of male rats (Rattus novergicus). The study involved 5 groups, P1 was the control group, while the other (P2-P5) were the case group with exposure of Pb in different concentration within 4 weeks. At the end of the exposure, the interaction between Pb and protein was determined using UV-Vis spectrophotometric method, and the Ca level was determined using permanganometric method. The results shows that that there is an interaction between Pb and bone protein. The result also shows that the value of the binding constant of Protein-Pb is 32.71. It means Pb have an high affinity to bind with bone protein, which promote a further reaction to induced the release of bone Ca from the bone protein. In conclusion, this present study found an obvious relationship between Pb and bone protein which promote a further reaction to increase the releasing of bone calcium.

  16. Denaturing high-performance liquid chromatography mutation analysis in patients with reduced Protein S levels

    DEFF Research Database (Denmark)

    Bathum, Lise; Münster, Anna-Marie; Nybo, Mads

    2008-01-01

    diagnosis and risk estimation. The aim was to design a high-throughput genetic analysis based on denaturing high-performance liquid chromatography to identify sequence variations in the gene coding for Protein S. PATIENTS: In total, 55 patients referred to the Section of Thrombosis and Haemostasis, Odense......BACKGROUND: Patients with congenital Protein S deficiency have increased risk of venous thromboembolism. However, Protein S levels show large intra-individual variation and the biochemical assays have low accuracy and a high interlaboratory variability. Genetic analysis might aid in a more precise......, giving a precise diagnosis and subsequently a better risk estimation....

  17. Serum eosinophil cationic protein levels can be useful for predicting acute exacerbation of asthma

    Directory of Open Access Journals (Sweden)

    Mitsuhiro Kamimura

    1998-01-01

    Full Text Available We report on a case in which five consecutive exacerbations of asthma were monitored by following serum eosinophil cationic protein (ECP levels. The serum ECP level correlated well with each exacerbation and tended to increase even before the exacerbations of asthma became apparent. This case shows that serum levels of ECP can be useful markers of disease activity and may also be predictive markers for acute exacerbation.

  18. Evolutionary tuning of protein expression levels of a positively autoregulated two-component system.

    Directory of Open Access Journals (Sweden)

    Rong Gao

    2013-10-01

    Full Text Available Cellular adaptation relies on the development of proper regulatory schemes for accurate control of gene expression levels in response to environmental cues. Over- or under-expression can lead to diminished cell fitness due to increased costs or insufficient benefits. Positive autoregulation is a common regulatory scheme that controls protein expression levels and gives rise to essential features in diverse signaling systems, yet its roles in cell fitness are less understood. It remains largely unknown how much protein expression is 'appropriate' for optimal cell fitness under specific extracellular conditions and how the dynamic environment shapes the regulatory scheme to reach appropriate expression levels. Here, we investigate the correlation of cell fitness and output response with protein expression levels of the E. coli PhoB/PhoR two-component system (TCS. In response to phosphate (Pi-depletion, the PhoB/PhoR system activates genes involved in phosphorus assimilation as well as genes encoding themselves, similarly to many other positively autoregulated TCSs. We developed a bacteria competition assay in continuous cultures and discovered that different Pi conditions have conflicting requirements of protein expression levels for optimal cell fitness. Pi-replete conditions favored cells with low levels of PhoB/PhoR while Pi-deplete conditions selected for cells with high levels of PhoB/PhoR. These two levels matched PhoB/PhoR concentrations achieved via positive autoregulation in wild-type cells under Pi-replete and -deplete conditions, respectively. The fitness optimum correlates with the wild-type expression level, above which the phosphorylation output saturates, thus further increase in expression presumably provides no additional benefits. Laboratory evolution experiments further indicate that cells with non-ideal protein levels can evolve toward the optimal levels with diverse mutational strategies. Our results suggest that the natural

  19. Ground level environmental protein concentrations in various ecuadorian environments: potential uses of aerosolized protein for ecological research

    Science.gov (United States)

    Staton, Sarah J.R.; Woodward, Andrea; Castillo, Josemar A.; Swing, Kelly; Hayes, Mark A.

    2014-01-01

    Large quantities of free protein in the environment and other bioaerosols are ubiquitous throughout terrestrial ground level environments and may be integrative indicators of ecosystem status. Samples of ground level bioaerosols were collected from various ecosystems throughout Ecuador, including pristine humid tropical forest (pristine), highly altered secondary humid tropical forest (highly altered), secondary transitional very humid forest (regrowth transitional), and suburban dry montane deforested (suburban deforested). The results explored the sensitivity of localized aerosol protein concentrations to spatial and temporal variations within ecosystems, and their value for assessing environmental change. Ecosystem specific variations in environmental protein concentrations were observed: pristine 0.32 ± 0.09 μg/m3, highly altered 0.07 ± 0.05 μg/m3, regrowth transitional 0.17 ± 0.06 μg/m3, and suburban deforested 0.09 ± 0.04 μg/m3. Additionally, comparisons of intra-environmental differences in seasonal/daily weather (dry season 0.08 ± 0.03 μg/m3 and wet season 0.10 ± 0.04 μg/m3), environmental fragmentation (buffered 0.19 ± 0.06 μg/m3 and edge 0.15 ± 0.06 μg/m3), and sampling height (ground level 0.32 ± 0.09 μg/m3 and 10 m 0.24 ± 0.04 μg/m3) demonstrated the sensitivity of protein concentrations to environmental conditions. Local protein concentrations in altered environments correlated well with satellite-based spectral indices describing vegetation productivity: normalized difference vegetation index (NDVI) (r2 = 0.801), net primary production (NPP) (r2 = 0.827), leaf area index (LAI) (r2 = 0.410). Moreover, protein concentrations distinguished the pristine site, which was not differentiated in spectral indices, potentially due to spectral saturation typical of highly vegetated environments. Bioaerosol concentrations represent an inexpensive method to increase understanding of environmental changes, especially in densely vegetated

  20. Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

    Science.gov (United States)

    Prabhu, Ashish A; Boro, Bibari; Bharali, Biju; Chakraborty, Shuchishloka; Dasu, V Venkata

    2018-03-28

    Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Despite of producing high protein titers, various cellular and process level bottlenecks hinders the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  1. Nitric Oxide Synthase and Cyclooxygenase Pathways: A Complex Interplay in Cellular Signaling.

    Science.gov (United States)

    Sorokin, Andrey

    2016-01-01

    The cellular reaction to external challenges is a tightly regulated process consisting of integrated processes mediated by a variety of signaling molecules, generated as a result of modulation of corresponding biosynthetic systems. Both, nitric oxide synthase (NOS) and cyclooxygenase (COX) systems, consist of constitutive forms (NOS1, NOS3 and COX-1), which are mostly involved in housekeeping tasks, and inducible forms (NOS2 and COX-2), which shape the cellular response to stress and variety of bioactive agents. The complex interplay between NOS and COX pathways can be observed at least at three levels. Firstly, products of NOS and Cox systems can mediate the regulation and the expression of inducible forms (NOS2 and COX-2) in response of similar and dissimilar stimulus. Secondly, the reciprocal modulation of cyclooxygenase activity by nitric oxide and NOS activity by prostaglandins at the posttranslational level has been shown to occur. Mechanisms by which nitric oxide can modulate prostaglandin synthesis include direct S-nitrosylation of COX and inactivation of prostaglandin I synthase by peroxynitrite, product of superoxide reaction with nitric oxide. Prostaglandins, conversely, can promote an increased association of dynein light chain (DLC) (also known as protein inhibitor of neuronal nitric oxide synthase) with NOS1, thereby reducing its activity. The third level of interplay is provided by intracellular crosstalk of signaling pathways stimulated by products of NOS and COX which contributes significantly to the complexity of cellular signaling. Since modulation of COX and NOS pathways was shown to be principally involved in a variety of pathological conditions, the dissection of their complex relationship is needed for better understanding of possible therapeutic strategies. This review focuses on implications of interplay between NOS and COX for cellular function and signal integration.

  2. Longevity in vivo of primary cell wall cellulose synthases.

    Science.gov (United States)

    Hill, Joseph Lee; Josephs, Cooper; Barnes, William J; Anderson, Charles T; Tien, Ming

    2018-02-01

    Our work focuses on understanding the lifetime and thus stability of the three main cellulose synthase (CESA) proteins involved in primary cell wall synthesis of Arabidopsis. It had long been thought that a major means of CESA regulation was via their rapid degradation. However, our studies here have uncovered that AtCESA proteins are not rapidly degraded. Rather, they persist for an extended time in the plant cell. Plant cellulose is synthesized by membrane-embedded cellulose synthase complexes (CSCs). The CSC is composed of cellulose synthases (CESAs), of which three distinct isozymes form the primary cell wall CSC and another set of three isozymes form the secondary cell wall CSC. We determined the stability over time of primary cell wall (PCW) CESAs in Arabidopsis thaliana seedlings, using immunoblotting after inhibiting protein synthesis with cycloheximide treatment. Our work reveals very slow turnover for the Arabidopsis PCW CESAs in vivo. Additionally, we show that the stability of all three CESAs within the PCW CSC is altered by mutations in individual CESAs, elevated temperature, and light conditions. Together, these results suggest that CESA proteins are very stable in vivo, but that their lifetimes can be modulated by intrinsic and environmental cues.

  3. Noise genetics: inferring protein function by correlating phenotype with protein levels and localization in individual human cells.

    Directory of Open Access Journals (Sweden)

    Shlomit Farkash-Amar

    2014-03-01

    Full Text Available To understand gene function, genetic analysis uses large perturbations such as gene deletion, knockdown or over-expression. Large perturbations have drawbacks: they move the cell far from its normal working point, and can thus be masked by off-target effects or compensation by other genes. Here, we offer a complementary approach, called noise genetics. We use natural cell-cell variations in protein level and localization, and correlate them to the natural variations of the phenotype of the same cells. Observing these variations is made possible by recent advances in dynamic proteomics that allow measuring proteins over time in individual living cells. Using motility of human cancer cells as a model system, and time-lapse microscopy on 566 fluorescently tagged proteins, we found 74 candidate motility genes whose level or localization strongly correlate with motility in individual cells. We recovered 30 known motility genes, and validated several novel ones by mild knockdown experiments. Noise genetics can complement standard genetics for a variety of phenotypes.

  4. The Common Inhalational Anesthetic Sevoflurane Induces Apoptosis and Increases β-Amyloid Protein Levels

    Science.gov (United States)

    Dong, Yuanlin; Zhang, Guohua; Zhang, Bin; Moir, Robert D.; Xia, Weiming; Marcantonio, Edward R.; Culley, Deborah J.; Crosby, Gregory; Tanzi, Rudolph E.; Xie, Zhongcong

    2009-01-01

    Objective: To assess the effects of sevoflurane, the most commonly used inhalation anesthetic, on apoptosis and β-amyloid protein (Aβ) levels in vitro and in vivo. Subjects: Naive mice, H4 human neuroglioma cells, and H4 human neuroglioma cells stably transfected to express full-length amyloid precursor protein. Interventions: Human H4 neuroglioma cells stably transfected to express full-length amyloid precursor protein were exposed to 4.1% sevoflurane for 6 hours. Mice received 2.5% sevoflurane for 2 hours. Caspase-3 activation, apoptosis, and Aβ levels were assessed. Results: Sevoflurane induced apoptosis and elevated levels of β-site amyloid precursor protein-cleaving enzyme and Aβ in vitro and in vivo. The caspase inhibitor Z-VAD decreased the effects of sevoflurane on apoptosis and Aβ. Sevoflurane-induced caspase-3 activation was attenuated by the γ-secretase inhibitor L-685,458 and was potentiated by Aβ. These results suggest that sevoflurane induces caspase activation which, in turn, enhances β-site amyloid precursor protein–cleaving enzyme and Aβ levels. Increased Aβ levels then induce further rounds of apoptosis. Conclusions: These results suggest that inhalational anesthetic sevoflurane may promote Alzheimer disease neuropathogenesis. If confirmed in human subjects, it may be prudent to caution against the use of sevoflurane as an anesthetic, especially in those suspected of possessing excessive levels of cerebral Aβ. PMID:19433662

  5. Elevated levels of the mismatch repair protein PMS2 are associated with prostate cancer.

    Science.gov (United States)

    Norris, Alixanna M; Woodruff, R D; D'Agostino, Ralph B; Clodfelter, Jill E; Scarpinato, Karin Drotschmann

    2007-02-01

    Defects in mismatch repair (MMR) proteins have been identified in various types of cancer. However, an association with prostate cancer has been controversial. Defective MMR results in genome instability with detrimental consequences that significantly contribute to tumorigenesis. This study determined alterations in key MMR protein levels in prostate cancer with the goal to identify prognostic markers. Prostatectomy samples were immunohistochemically stained and the relative presence or absence of key proteins MSH2, MLH1, and PMS2 determined. Cancer tissue of distinct grades was compared with the normal surrounding tissue. Microsatellite instability (MSI) in altered tissues was determined according to NCI guidelines. In contrast to reports that associate a lack of individual MMR proteins with tumorigenesis, a significant increase in PMS2 levels was identified in PIN lesions and prostate cancer tissue. This elevation in PMS2 was independent of changes in levels in its heterodimeric partner, MLH1. Prostate tumors with elevated levels of PMS2 were genetically unstable, which was corrected by MLH1 co-elevation. This is the first documentation of detrimental consequences associated with the increase in a MMR protein in human cancer. This study recognizes PMS2 elevation as a prognostic marker in pre-neoplastic and prostate cancer lesions. This result has significant implications for future diagnostic and treatment measures. (c) 2006 Wiley-Liss, Inc.

  6. CTP limitation increases expression of CTP synthase in Lactococcus lactis

    DEFF Research Database (Denmark)

    Jørgensen, C.M.; Hammer, Karin; Martinussen, Jan

    2003-01-01

    CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which beta-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold...... decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced level. CTP limitation has pronounced effects on central cellular metabolism, and both RNA and protein...... for regulation of the pyrG gene. It is possible to fold the pyrG leader in an alternative structure that would prevent the formation of the terminator. We suggest a model for pyrG regulation in L. lactis, and probably in other gram-positive bacteria as well, in which pyrG expression is directly dependent...

  7. In vitro effect of dietary protein level and nondigestible oligosaccharides on feline fecal microbiota.

    Science.gov (United States)

    Pinna, C; Stefanelli, C; Biagi, G

    2014-12-01

    The aim of the present study was to evaluate in vitro the effect of some prebiotic substances and 2 dietary protein levels on the composition and activity of feline fecal microbiota. Two in vitro studies were conducted. First, 6 nondigestible oligosaccharides were studied; treatments were control diet (CTRL), gluconic acid (GA), carrot fiber (CF), fructooligosaccharides (FOS), galactooligosaccharides (GOS), lactitol (LAC), and pectins from citrus fruit (PEC). Substrates were added to feline fecal cultures at 2 g/L for 24 h incubation. Compared with the CTRL, ammonia had been reduced (Pmicrobiota and that high dietary protein levels in a cat's diet can have negative effects on the animal intestinal environment.

  8. Neuronal Nitric Oxide Synthase Induction in the Antitumorigenic and Neurotoxic Effects of 2-Methoxyestradiol

    Directory of Open Access Journals (Sweden)

    Magdalena Gorska

    2014-08-01

    Full Text Available Objective: 2-Methoxyestradiol, one of the natural 17β-estradiol derivatives, is a novel, potent anticancer agent currently being evaluated in advanced phases of clinical trials. The main goal of the study was to investigate the anticancer activity of 2-methoxy-estradiol towards osteosarcoma cells and its possible neurodegenerative effects. We used an experimental model of neurotoxicity and anticancer activity of the physiological agent, 2-methoxyestradiol. Thus, we used highly metastatic osteosarcoma 143B and mouse immortalized hippocampal HT22 cell lines. The cells were treated with pharmacological (1 μM, 10 μM concentrations of 2-methoxyestradiol. Experimental: Neuronal nitric oxide synthase and 3-nitrotyrosine protein levels were determined by western blotting. Cell viability and induction of cell death were measured by MTT and PI/Annexin V staining and a DNA fragmentation ELISA kit, respectively. Intracellular levels of nitric oxide were determined by flow cytometry. Results: Here we demonstrated that the signaling pathways of neurodegenerative diseases and cancer may overlap. We presented evidence that 2-methoxyestradiol, in contrast to 17β-estradiol, specifically affects neuronal nitric oxide synthase and augments 3-nitrotyrosine level leading to osteosarcoma and immortalized hippocampal cell death. Conclusions: We report the dual facets of 2-methoxyestradiol, that causes cancer cell death, but on the other hand may play a key role as a neurotoxin.

  9. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    Science.gov (United States)

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. The effects of maternal total protein, albumin and hemoglobin levels on birth weight

    Directory of Open Access Journals (Sweden)

    Berna Haliloglu

    2007-12-01

    Full Text Available OBJECTIVE: The present study was designed to investigate the influence of third trimester maternal total protein, albumin, hemoglobin levels on birth weight.\tMATERIAL-METHOD: Between January 2005 and July 2005, 750 pregnant women applied for delivery at Zeynep Kamil Women’s and Children Education and Research Hospital at 37-40 week’s gestation were examined. Maternal total protein, albumin and hemoglobin levels were measured. Data included maternal age, gravidity, parity, gestational age, birth weight, gender, presence of iron supplementation and its duration.\tRESULTS: The birth weight was significantly higher in anemic and hypoproteinemic groups compared those with normal levels. After adjusting for counfounding factors, significance of both findings lost. The cases received iron supplementation had infants with higher birth weight, however, it was not statistically significant (p: 0.055. A significant positive relation was observed between birth weight and maternal age, gravidity, parity and gestational age. No relation found between maternal total protein, albumin, hemoglobin levels and birth weight.\tCONCLUSION: The last trimester maternal total protein, albumin, hemoglobin levels seem not to be a determining factor on infant's birth weight.

  11. Investigation of the molecular level interactions between mucins and food proteins: Spectroscopic, tribological and rheological studies

    DEFF Research Database (Denmark)

    Celebioglu, Hilal Yilmaz

    The thesis investigated the structure and molecular-level interaction of β-lactoglobulin (BLG) and mucins, representing major components of the dairy products and saliva/digestion systems, respectively. Mucins are long glycoprotein molecules responsible for the gel nature of the mucous layer covers...... epithelial surfaces throughout the body. A literature review of the interactions of different mucin types and saliva mucins with several food proteins and food protein emulsions, as well as their functional properties related to the food oral processing is presented at the first chapter of the thesis (Paper...... V). Most of the studies suggest an electrostatic attraction between positively charged food proteins with negatively charged moieties of mucins (mainly on glycosylated region of mucins). The structural changes occurring during the interaction between BLG, the major whey protein, and bovine...

  12. Effect of voluntary exercise and dietary protein levels on incorporation of 14C-leucine into protein by mice liver slices in vitro

    International Nuclear Information System (INIS)

    Yashiro, Masanori; Kimura, Shuichi

    1983-01-01

    The effect of voluntary exercise on incorporation of 14 C-leucine into protein by mice liver slices in vitro were examined with mice fed 4 %, 6 % and 20 % protein diets. The incorporation of 14 C-leucine increased as dietary protein levels decreased and was significantly higher in liver slices of exercise groups than in slices of non-exercise groups. (author)

  13. The subcellular localization of yeast glycogen synthase is dependent upon glycogen content

    OpenAIRE

    Wilson, Wayne A.; Boyer, Michael P.; Davis, Keri D.; Burke, Michael; Roach, Peter J.

    2010-01-01

    The budding yeast, Saccharomyces cerevisiae, accumulates the storage polysaccharide glycogen in response to nutrient limitation. Glycogen synthase, the major form of which is encoded by the GSY2 gene, catalyzes the key regulated step in glycogen storage. Here, we utilize Gsy2p fusions to green fluorescent protein (GFP) to determine where glycogen synthase is located within cells. We demonstrate that the localization pattern of Gsy2-GFP depends upon the glycogen content of the cell. When glyco...

  14. Effects of dietary protein level on nutrients digestibility and reproductive performance of female mink (Neovison vison during gestation

    Directory of Open Access Journals (Sweden)

    Qingkui Jiang

    2015-06-01

    Full Text Available The objective of this study was to determine whether nutrient digestibility and reproductive performance of pregnant mink (Neovison vison were affected by different dietary protein levels. One hundred and twenty female mink were randomly assigned to four groups, receiving diets of fresh material with different protein levels. The dietary protein levels, expressed as percentage of dry matter (DM, were 32, 36, 40 and 44% respectively. These values corresponded to average 320, 360, 400 and 440 g protein/kg DM, respectively. Results were as follows. All of crude protein digestibility, nitrogen (N intake, N retention increased along with dietary protein level increasing. Low protein level (32% significantly reduced the above indicators (P < 0.05. DM digestibility and ether extract digestibility were not affected by dietary protein level. Results of mated females, barren females, kids per litter, live born kids per mated female, birth survival rate, and birth weight showed that mink achieved optimal reproductive performance when dietary protein level was 36%. In conclusion, dietary protein was anticipated to significantly influence some nutrients' utilization. Adopting the appropriate dietary protein level allow better reproduction performance. The most preferable reproductive performance was achieved when diet contained 275.5 g digestible protein per kg DM for female mink in gestation.

  15. A fish protein hydrolysate alters fatty acid composition in liver and adipose tissue and increases plasma carnitine levels in a mouse model of chronic inflammation.

    Science.gov (United States)

    Bjørndal, Bodil; Berge, Christ; Ramsvik, Marie Sannes; Svardal, Asbjørn; Bohov, Pavol; Skorve, Jon; Berge, Rolf K

    2013-10-07

    There is growing evidence that fish protein hydrolysate (FPH) diets affect mitochondrial fatty acid metabolism in animals. The aim of the study was to determine if FPH could influence fatty acid metabolism and inflammation in transgene mice expressing human tumor necrosis factor alpha (hTNFα). hTNFα mice (C57BL/6 hTNFα) were given a high-fat (23%, w/w) diet containing 20% casein (control group) or 15% FPH and 5% casein (FPH group) for two weeks. After an overnight fast, blood, adipose tissue, and liver samples were collected. Gene expression and enzyme activity was analysed in liver, fatty acid composition was analyzed in liver and ovarian white adipose tissue, and inflammatory parameters, carnitine, and acylcarnitines were analyzed in plasma. The n-3/n-6 fatty acid ratio was higher in mice fed the FPH diet than in mice fed the control diet in both adipose tissue and liver, and the FPH diet affected the gene expression of ∆6 and ∆9 desaturases. Mice fed this diet also demonstrated lower hepatic activity of fatty acid synthase. Concomitantly, a lower plasma INF-γ level was observed. Plasma carnitine and the carnitine precursor γ-butyrobetaine was higher in the FPH-group compared to control, as was plasma short-chained and medium-chained acylcarnitine esters. The higher level of plasma acetylcarnitine may reflect a stimulated mitochondrial and peroxisomal β-oxidation of fatty acids, as the hepatic activities of peroxisomal acyl-CoA oxidase 1 and mitochondrial carnitine palmitoyltransferase-II were higher in the FPH-fed mice. The FPH diet was shown to influence hepatic fatty acid metabolism and fatty acid composition. This indicates that effects on fatty acid metabolism are important for the bioactivity of protein hydrolysates of marine origin.

  16. Effects of nutritional supplementation on periodontal parameters, carotenoid antioxidant levels, and serum C-reactive protein.

    Science.gov (United States)

    Harpenau, Lisa A; Cheema, Abida T; Zingale, Joseph A; Chambers, David W; Lundergan, William P

    2011-05-01

    Few studies have focused on the role of nutrition in periodontal disease. The purpose of this trial was to determine the effect of a nutritional supplement on gingival inflammation, bleeding, probing depth, clinical attachment level, carotenoid antioxidant level, and C-reactive protein. The test supplement, consisting of a standard multivitamin formula, as well as several phytonutrients associated with antiinflammatory/antioxidant effects, provided modest benefits in reducing inflammation; however, further studies with larger populations and longer intervention are warranted.

  17. Effects of energy and protein levels on growth and nutrient utilization ...

    African Journals Online (AJOL)

    Effects of energy and protein levels on growth and nutrient utilization of weaned rabbits. ML Egbo, TA Adegbola, EO Oyawoye, MM Abubakar. Abstract. No Abstract. Animal Production Research Advances Vol. 3 (4) 2007: pp. 306-310. http://dx.doi.org/10.4314/apra.v3i4.36411 · AJOL African Journals Online. HOW TO USE ...

  18. Levels of ABA, its precursors and dehydrin-like proteins during ...

    African Journals Online (AJOL)

    2Department of Molecular Biology and Biotechnology, University of Dar es Salaam,. P.O Box 35179, Dar ... to combat stress. Levels of ABA and proteins that cross reacted with an anti – dehydrin ...... Cambridge, Melbourne). Wang, X.-Q., Ullah ...

  19. Systemic Glucose Level Changes with a Carbohydrate-Restricted and Higher Protein Diet Combined with Exercise

    Science.gov (United States)

    Bowden, Rodney G.; Lanning, Beth A.; Doyle, Eva I.; Slonaker, Becky; Johnston, Holly M.; Scanes, Georgene

    2007-01-01

    Objective: The authors' purpose in this study was to compare the effects of macronutrient intake on systemic glucose levels in previously sedentary participants who followed 1 of 4 diets that were either higher protein or high carbohydrate, while initiating an exercise program. Participants and Methods: The authors randomly assigned 94 sedentary…

  20. Altered protein and iron levels of patients with active tuberculosis in ...

    African Journals Online (AJOL)

    Backgound: Tuberculosis as a state of chronic inflammation impacts on haematologic functions of the body. Objectives: This study aimed at assessing iron parameters and serum protein levels of ninety tuberculosis patients aged fifteen to sixty years, enrolled from Dr Lawrence Henshaw Memorial Hospital, Calabar, Nigeria.

  1. CSF Neurofilament Proteins Levels are Elevated in Sporadic Creutzfeldt-Jakob Disease

    NARCIS (Netherlands)

    van Eijk, Jeroen J. J.; van Everbroeck, Bart; Abdo, W. Farid; Kremer, Berry P. H.; Verbeek, Marcel M.

    2010-01-01

    In this study we investigated the cerebrospinal fluid (CSF) levels of neurofilament light (NFL) and heavy chain (NFHp35), total tau (t-tau), and glial fibrillary acidic protein (GFAP) to detect disease specific profiles in sporadic Creutzfeldt Jakob disease (sCJD) patients and Alzheimer's disease

  2. Modeling of DNA and Protein Organization Levels with Cn3D Software

    Science.gov (United States)

    Stasinakis, Panagiotis K.; Nicolaou, Despoina

    2017-01-01

    The molecular structure of living organisms and the complex interactions amongst its components are the basis for the diversity observed at the macroscopic level. Proteins and nucleic acids are some of the major molecular components, and play a key role in several biological functions, such as those of development and evolution. This article…

  3. Plasma levels of C-Reactive Protein and Fibrinogen in Pulmonary ...

    African Journals Online (AJOL)

    In this study, we determined the changes in plasma C- reactive protein (C-RP) and Fibrinogen levels in Drug sensitive Tuberculosis (DSTB) patients at diagnosis, Multi drug resistant tuberculosis (MDRTB) patients at diagnosis and during chemotherapy. Twenty-four (24) patients MDRTB patients and 24 newly diagnosed ...

  4. Protein C and antithrombin levels in patients with sickle cell anemia ...

    African Journals Online (AJOL)

    Background: Alterations in the components of hemostasis, namely platelet function, the procoagulant, anticoagulant, and the fibrinolytic systems, are observed in sickle cell anemia (SCA) and are in favor of a procoagulant phenotype. Therefore, study of protein C and antithrombin (AT) levels in patients with SCA in steady ...

  5. Flour sodium dodecyl sulfate (SDS)-extractable protein level as a cookie flour quality indicator.

    Science.gov (United States)

    Pareyt, Bram; Bruneel, Charlotte; Brijs, Kristof; Goesaert, Hans; Delcour, Jan A

    2010-01-13

    Flour characteristics of laboratory-milled flour fractions of two wheat cultivars were related to their cookie-baking performance. Cultivar (cv.) Albatros wheat milling yielded fractions with lower damaged starch (DS) and arabinoxylan levels and higher sodium dodecyl sulfate-extractable protein (SDSEP) levels than did cv. Meunier wheat milling. During baking, cv. Albatros flour doughs spread faster and set later than their cv. Meunier counterparts and, hence, resulted in larger cookie diameters. DS levels negatively affected spread rate during both cv. Albatros (R2=0.68) and cv. Meunier (R2=0.51) cookie baking. SDSEP levels also influenced cookie quality. The use of flour heat-treated to reduce its SDSEP levels to different degrees led to reduction of the set time (R2=0.90). It was deduced that larger gluten polymer sizes limit dough spread time during baking and that, apart from DS level, the SDSEP level is an indicator for cookie flour quality.

  6. Comparison of C-reactive protein and high-sensitivity C-reactive protein levels in patients on hemodialysis

    Directory of Open Access Journals (Sweden)

    Imed Helal

    2012-01-01

    Full Text Available Chronic inflammation is highly prevalent in patients on hemodialysis (HD, as evidenced by increased levels of C-reactive protein (CRP. We compared CRP to high-sensitivity C-reactive protein (hs-CRP to determine whether it has any clinical implications and prognostic significance in terms of mortality. CRP was measured using a standard immunoturbidometric assay on the COBAS; INTEGRA system and hs-CRP was measured using the Dade Behring on the Konelab Nephelometer in 50 patients on HD. CRP (≥6 mg/L and hs-CRP (≥3 mg/L levels were elevated in 30% and 54% of the patients, respectively. A significant correlation was noted between hs-CRP and CRP levels (r = 0.98, P <0.001. Deming regression analysis showed that the slope was near one (r = 0.90; 0.83-0.94 and that the intercept was small. Multivariate regression confirmed that age above 40 years (RR = 3.69, P = 0.027 and duration on HD greater than five years (RR = 3.71, P = 0.028 remained significant independent predictors of serum hs-CRP. Thirteen patients died during follow-up (26%. Multivariate Cox regression demonstrated that hs-CRP (RR = 1.062, P = 0.03 and CRP levels (RR = 1.057, P = 0.009 and age (RR = 1.078, P = 0.001 were the most powerful predictors of mortality. The CRP standard assay presents a reasonable alternative to the hs-CRP assay in patients on HD. The advantages of the CRP standard assay are its online and real-time availability as well as lower costs, particularly in developing countries.

  7. Reduced methylation of the thromboxane synthase gene is correlated with its increased vascular expression in preeclampsia.

    Science.gov (United States)

    Mousa, Ahmad A; Strauss, Jerome F; Walsh, Scott W

    2012-06-01

    Preeclampsia is characterized by increased thromboxane and decreased prostacyclin levels, which predate symptoms, and can explain some of the clinical manifestations of preeclampsia, including hypertension and thrombosis. In this study, we examined DNA methylation of the promoter region of the thromboxane synthase gene (TBXAS1) and the expression of thromboxane synthase in systemic blood vessels of normal pregnant and preeclamptic women. Thromboxane synthase is responsible for the synthesis of thromboxane A(2), a potent vasoconstrictor and activator of platelets. We also examined the effect of experimentally induced DNA hypomethylation on the expression of thromboxane synthase in a neutrophil-like cell line (HL-60 cells) and in cultured vascular smooth muscle and endothelial cells. We found that DNA methylation of the TBXAS1 promoter was decreased and thromboxane synthase expression was increased in omental arteries of preeclamptic women as compared with normal pregnant women. Increased thromboxane synthase expression was observed in vascular smooth muscles cells, endothelial cells, and infiltrating neutrophils. Experimentally induced DNA hypomethylation only increased expression of thromboxane synthase in the neutrophil-like cell line, whereas tumor necrosis factor-α, a neutrophil product, increased its expression in cultured vascular smooth muscle cells. Our study suggests that epigenetic mechanisms and release of tumor necrosis factor-α by infiltrating neutrophils could contribute to the increased expression of thromboxane synthase in maternal systemic blood vessels, contributing to the hypertension and coagulation abnormalities associated with preeclampsia.

  8. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    OpenAIRE

    Karuppiah, Vijaykumar; Ranaghan, Kara E.; Leferink, Nicole G. H.; Johannissen, Linus O.; Shanmugam, Muralidharan; Ní Cheallaigh, Aisling; Bennett, Nathan J.; Kearsey, Lewis J.; Takano, Eriko; Gardiner, John M.; van der Kamp, Marc W.; Hay, Sam; Mulholland, Adrian J.; Leys, David; Scrutton, Nigel S.

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS)...

  9. Endostatin gene variation and protein levels in breast cancer susceptibility and severity

    International Nuclear Information System (INIS)

    Balasubramanian, Sabapathy P; Cross, Simon S; Globe, Jenny; Cox, Angela; Brown, Nicola J; Reed, Malcolm W

    2007-01-01

    Endostatin is a potent endogenous anti-angiogenic agent which inhibits tumour growth. A non-synonymous coding polymorphism in the Endostatin gene is thought to affect Endostatin activity. We aimed to determine the role of this Endostatin polymorphism in breast cancer pathogenesis and any influence on serum Endostatin levels in healthy volunteers. Endostatin protein expression on a breast cancer micro array was also studied to determine any relationship to genotype and to breast cancer prognosis. The 4349G > A (coding non-synonymous) polymorphism in exon 42 of the Endostatin gene was genotyped in approximately 846 breast cancer cases and 707 appropriate controls. In a separate healthy cohort of 57 individuals, in addition to genotyping, serum Endostatin levels were measured using enzyme linked immunosorbant assay (ELISA). A semi-quantitative assessment of Endostatin protein expression on immunostained tissue micro arrays (TMA) constructed from breast cancer samples of patients with genotype data was performed. The rare allele (A) was significantly associated with invasive breast cancers compared to non-invasive tumours (p = 0.03), but there was no association with tumour grade, nodal status, vascular invasion or overall survival. There was no association with breast cancer susceptibility. Serum Endostatin levels and Endostatin protein expression on the tissue micro array were not associated with genotype. The Endostatin 4349A allele is associated with invasive breast cancer. The Endostatin 4349G > A polymorphism however does not appear to be associated with breast cancer susceptibility or severity in invasive disease. By studying circulating levels and tumour Endostatin protein expression, we have shown that any influence of this polymorphism is unlikely to be through an effect on the levels of protein produced

  10. myo-Inositol-1-phosphate synthase is required for polar auxin transport and organ development

    KAUST Repository

    Chen, Hao

    2010-06-01

    myo-Inositol-1-phosphate synthase is a conserved enzyme that catalyzes the first committed and rate-limiting step in inositol biosynthesis. Despite its wide occurrence in all eukaryotes, the role of myo-inositol-1-phosphate synthase and de novo inositol biosynthesis in cell signaling and organism development has been unclear. In this study, we isolated loss-of-function mutants in the Arabidopsis MIPS1 gene from different ecotypes. It was found that all mips1 mutants are defective in embryogenesis, cotyledon venation patterning, root growth, and root cap development. The mutant roots are also agravitropic and have reduced basipetal auxin transport. mips1 mutants have significantly reduced levels of major phosphatidylinositols and exhibit much slower rates of endocytosis. Treatment with brefeldin A induces slower PIN2 protein aggregation in mips1, indicating altered PIN2 trafficking. Our results demonstrate that MIPS1 is critical for maintaining phosphatidylinositol levels and affects pattern formation in plants likely through regulation of auxin distribution. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Amelioration of ultraviolet-B-induced down-regulation of mRNA levels for chloroplast proteins, by high irradiance, is mediated by photosynthesis

    International Nuclear Information System (INIS)

    Mackerness, S.A. H.; Butt, P.J.; Jordan, B.R.; Thomas, B.

    1996-01-01

    The mechanism by which increasing photosynthetically active radiation (PAR) reduces the sensitivity of RNA transcripts to UV-B radiation was studied in pea (Pisum sativum L.). mRNA transcript levels for rbc S, rbc L, cab and psb A were measured over an 8 d experimental period in pea, plants supplemented with UV-B radiation under a range of conditions. Under low light (150 mu-mol m -2 s -1 ), UV-B resulted in a significant decline in the levels of transcripts for all four genes which was prevented by increasing the background irradiance to 350 mu-mol m -2 s -1 (high light) with white light from fluorescent lamps. Increasing CO 2 levels to give photosynthesis rates equivalent to the high light treatment partially protected rbc S and cab transcripts and fully protected rbc L transcripts but did not prevent visible injury. Increasing light with low pressure sodium lamps, which increase photosynthesis but are not effective for activation of the DNA repair enzyme, photolyase, gave results which were not significantly different from white fluorescent high light treatments. Protection by high light was lost in the presence of the photosynthesis inhibitors CCCP and DCMU. The UV-B induced increase in the expression of chalcone synthase (chs) genes was delayed by the treatments which increased photosynthesis rates and conferred protection. The results indicate that photosynthesis plays a key role in the amelioration of UV-B induced decline in mRNA levels for proteins. The minimal role of DNA repair by photolyase indicates that reduction in photosynthesis gene transcripts in response to UV-B represents a specific regulation rather than being a consequence of DNA damage. (author)

  12. Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

    Science.gov (United States)

    Liang, Xue-Hai; Sun, Hong; Shen, Wen; Wang, Shiyu; Yao, Joyee; Migawa, Michael T; Bui, Huynh-Hoa; Damle, Sagar S; Riney, Stan; Graham, Mark J; Crooke, Rosanne M; Crooke, Stanley T

    2017-09-19

    A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Levels of digestible protein to surubim (Pseudoplatystoma sp. reared in net cages

    Directory of Open Access Journals (Sweden)

    Claucia Aparecida Honorato

    2014-10-01

    Full Text Available The Pseudoplatystoma sp. is species of carnivorous fish that require special attention in the diet offered. This work had the objective to determine the digestible protein requirement of juvenile the Pseudoplatystoma sp. reared in net cages. The test consisted of four isoenergetic diets (2606.69 ± 39.16 kcal kg-1 of digestible energy containing increasing levels of digestible protein (23, 24, 26 and 28%PD provided to juveniles of surubim (157.35±11.23g for five months. The parameters of growth, fillet composition, metabolic liver enzymes and morphometry of the intestine and liver were analyzed in completely randomized design with four treatments and four replicates. An increase of protein in the diet provided better weight gain. The metabolic liver enzymes increased in fish fed 24PD. The histopathological changes were not observed in the liver of the fish. The bowel histology showed adaptation to increased protein in the diet until the 26 level PD. juveniles of Pseudoplatystoma sp. Were demanding in digestible protein, showing the best results of production performance and nutrient use efficiency with the diet containing 28%PD.

  14. Human METTL12 is a mitochondrial methyltransferase that modifies citrate synthase.

    Science.gov (United States)

    Rhein, Virginie F; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-06-01

    The protein methylome in mammalian mitochondria has been little studied until recently. Here, we describe that lysine-368 of human citrate synthase is methylated and that the modifying enzyme, localized in the mitochondrial matrix, is methyltransferase-like protein 12 (METTL12), a member of the family of 7β-strand methyltransferases. Lysine-368 is near the active site of citrate synthase, but removal of methylation has no effect on its activity. In mitochondria, it is possible that some or all of the enzymes of the citric acid cycle, including citrate synthase, are organized in metabolons to facilitate the channelling of substrates between participating enzymes. Thus, possible roles for the methylation of Lys-368 are in controlling substrate channelling itself, or in influencing protein-protein interactions in the metabolon. © 2017 The Authors FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  15. Trace levels of innate immune response modulating impurities (IIRMIs) synergize to break tolerance to therapeutic proteins.

    Science.gov (United States)

    Verthelyi, Daniela; Wang, Vivian

    2010-12-22

    Therapeutic proteins such as monoclonal antibodies, replacement enzymes and toxins have significantly improved the therapeutic options for multiple diseases, including cancer and inflammatory diseases as well as enzyme deficiencies and inborn errors of metabolism. However, immune responses to these products are frequent and can seriously impact their safety and efficacy. Of the many factors that can impact protein immunogenicity, this study focuses on the role of innate immune response modulating impurities (IIRMIs) that could be present despite product purification and whether these impurities can synergize to facilitate an immunogenic response to therapeutic proteins. Using lipopolysaccharide (LPS) and CpG ODN as IIRMIs we showed that trace levels of these impurities synergized to induce IgM, IFNγ, TNFα and IL-6 expression. In vivo, trace levels of these impurities synergized to increase antigen-specific IgG antibodies to ovalbumin. Further, whereas mice treated with human erythropoietin showed a transient increase in hematocrit, those that received human erythropoietin containing low levels of IIRMIs had reduced response to erythropoietin after the 1(st) dose and developed long-lasting anemia following subsequent doses. This suggests that the presence of IIRMIs facilitated a breach in tolerance to the endogenous mouse erythropoietin. Overall, these studies indicate that the risk of enhancing immunogenicity should be considered when establishing acceptance limits of IIRMIs for therapeutic proteins.

  16. Protein logic: a statistical mechanical study of signal integration at the single-molecule level.

    Science.gov (United States)

    de Ronde, Wiet; Rein ten Wolde, Pieter; Mugler, Andrew

    2012-09-05

    Information processing and decision-making is based upon logic operations, which in cellular networks has been well characterized at the level of transcription. In recent years, however, both experimentalists and theorists have begun to appreciate that cellular decision-making can also be performed at the level of a single protein, giving rise to the notion of protein logic. Here we systematically explore protein logic using a well-known statistical mechanical model. As an example system, we focus on receptors that bind either one or two ligands, and their associated dimers. Notably, we find that a single heterodimer can realize any of the 16 possible logic gates, including the XOR gate, by variation of biochemical parameters. We then introduce what to our knowledge is a novel idea: that a set of receptors with fixed parameters can encode functionally unique logic gates simply by forming different dimeric combinations. An exhaustive search reveals that the simplest set of receptors (two single-ligand receptors and one double-ligand receptor) can realize several different groups of three unique gates, a result for which the parametric analysis of single receptors and dimers provides a clear interpretation. Both results underscore the surprising functional freedom readily available to cells at the single-protein level. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. Trace levels of innate immune response modulating impurities (IIRMIs synergize to break tolerance to therapeutic proteins.

    Directory of Open Access Journals (Sweden)

    Daniela Verthelyi

    Full Text Available Therapeutic proteins such as monoclonal antibodies, replacement enzymes and toxins have significantly improved the therapeutic options for multiple diseases, including cancer and inflammatory diseases as well as enzyme deficiencies and inborn errors of metabolism. However, immune responses to these products are frequent and can seriously impact their safety and efficacy. Of the many factors that can impact protein immunogenicity, this study focuses on the role of innate immune response modulating impurities (IIRMIs that could be present despite product purification and whether these impurities can synergize to facilitate an immunogenic response to therapeutic proteins. Using lipopolysaccharide (LPS and CpG ODN as IIRMIs we showed that trace levels of these impurities synergized to induce IgM, IFNγ, TNFα and IL-6 expression. In vivo, trace levels of these impurities synergized to increase antigen-specific IgG antibodies to ovalbumin. Further, whereas mice treated with human erythropoietin showed a transient increase in hematocrit, those that received human erythropoietin containing low levels of IIRMIs had reduced response to erythropoietin after the 1(st dose and developed long-lasting anemia following subsequent doses. This suggests that the presence of IIRMIs facilitated a breach in tolerance to the endogenous mouse erythropoietin. Overall, these studies indicate that the risk of enhancing immunogenicity should be considered when establishing acceptance limits of IIRMIs for therapeutic proteins.

  18. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

    Directory of Open Access Journals (Sweden)

    Meili Gao

    2012-01-01

    Full Text Available Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed.

  19. Endothelial nitric oxide synthase deficiency influences normal cell cycle progression and apoptosis in trabecular meshwork cells

    Directory of Open Access Journals (Sweden)

    Qiong Liao

    2016-06-01

    Full Text Available AIM: To clarify how the endothelial nitric oxide synthase (eNOS, NOS3 make effect on outflow facility through the trabecular meshwork (TM. METHODS: Inhibition of NOS3 gene expression in human TM cells were conducted by three siRNAs. Then the mRNA and protein levels of NOS3 in siRNA-treated and negative control (NC cells were determined, still were the collagen, type IV, alpha 1 (COL4A1 and fibronectin 1 by real-time PCR and Western blot analysis. In addition, NOS3 concentrations in culture supernatant fluids of TM cells were measured. Cell cycle and cell apoptosis analysis were performed using flow cytometry. RESULTS: The mRNA level of NOS3 was decreased by three different siRNA interference, similar results were obtained not only of the relative levels of NOS3 protein, but also the expression levels of COL4A1 and fibronectin 1. The number of cells in S phase was decreased, while contrary result was obtained in G2 phase. The number of apoptotic cells in siRNA-treated groups were significant increased compared to the NC samples. CONCLUSION: Abnormal NOS3 expression can make effects on the proteins levels of extracellular matrix component (e.g. fibronectin 1 and COL4A1. Reduced NOS3 restrains the TM cell cycle progression at the G2/M-phase transition and induced cell apoptosis.

  20. Anti-carbamylated Protein Antibody Levels Correlate with Anti-Sa (Citrullinated Vimentin) Antibody Levels in Rheumatoid Arthritis.

    Science.gov (United States)

    Challener, Gregory J; Jones, Jonathan D; Pelzek, Adam J; Hamilton, B JoNell; Boire, Gilles; de Brum-Fernandes, Artur José; Masetto, Ariel; Carrier, Nathalie; Ménard, Henri A; Silverman, Gregg J; Rigby, William F C

    2016-02-01

    The presence of anticitrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) indicates a breach in immune tolerance. Recent studies indicate that this breach extends to homocitrullination of lysines with the formation of anti-carbamylated protein (anti-CarP) antibodies. We analyzed the clinical and serologic relationships of anti-CarP in 2 RA cohorts. Circulating levels of immunoglobulin G anti-CarP antibodies were determined by ELISA in established (Dartmouth-Hitchcock Medical Center) and early (Sherbrooke University Hospital Center) cohorts and evaluated for anticyclic citrullinated peptide antibodies (anti-CCP), specific ACPA, and rheumatoid factor (RF) levels using the Student t test and correlation analysis. We identified elevated anti-CarP antibodies titers in 47.0% of seropositive patients (Dartmouth, n = 164), with relationships to anti-CCP (p < 0.0001) and IgM-RF (p = 0.001). Similarly, 38.2% of seropositive patients from the Sherbrooke cohort (n = 171) had elevated anti-CarP antibodies; titers correlated to anti-CCP (p = 0.01) but not IgM-RF (p = 0.09). A strong correlation with anti-Sa was observed: 47.9% anti-Sa+ patients were anti-CarP antibodies+ versus only 25.4% anti-Sa- in the Sherbrooke cohort (p = 0.0002), and 62.6% anti-Sa+ patients versus 26.9% anti-Sa- were anti-CarP antibodies+ in Dartmouth (p < 0.0001). We found a more variable response for reactivity to citrullinated fibrinogen or to citrullinated peptides from fibrinogen and α enolase. In 2 North American RA cohorts, we observed a high prevalence of anti-CarP antibody positivity. We also describe a surprising and unexpected association of anti-CarP with anti-Sa antibodies that could not be explained by cross-reactivity. Further, considerable heterogeneity exists between anti-CarP reactivity and other citrullinated peptide reactivity, raising the question of how the pathogenesis of antibody responses for carbamylated proteins and citrullinated proteins may be linked in vivo.

  1. Soybean seeds expressing feedback-insensitive cystathionine γ-synthase exhibit a higher content of methionine.

    Science.gov (United States)

    Song, Shikui; Hou, Wensheng; Godo, Itamar; Wu, Cunxiang; Yu, Yang; Matityahu, Ifat; Hacham, Yael; Sun, Shi; Han, Tianfu; Amir, Rachel

    2013-04-01

    Soybean seeds provide an excellent source of protein for human and livestock nutrition. However, their nutritional quality is hampered by a low concentration of the essential sulfur amino acid, methionine (Met). In order to study factors that regulate Met synthesis in soybean seeds, this study used the Met-insensitive form of Arabidopsis cystathionine γ-synthase (AtD-CGS), which is the first committed enzyme of Met biosynthesis. This gene was expressed under the control of a seed-specific promoter, legumin B4, and used to transform the soybean cultivar Zigongdongdou (ZD). In three transgenic lines that exhibited the highest expression level of AtD-CGS, the level of soluble Met increased significantly in developing green seeds (3.8-7-fold). These seeds also showed high levels of other amino acids. This phenomenon was more prominent in two transgenic lines, ZD24 and ZD91. The total Met content, which including Met incorporated into proteins, significantly increased in the mature dry seeds of these two transgenic lines by 1.8- and 2.3-fold, respectively. This elevation was accompanied by a higher content of other protein-incorporated amino acids, which led to significantly higher total protein content in the seeds of these two lines. However, in a third transgenic line, ZD01, the level of total Met and the level of other amino acids did not increase significantly in the mature dry seeds. This line also showed no significant change in protein levels. This suggests a positive connection between high Met content and the synthesis of other amino acids that enable the synthesis of more seed proteins.

  2. Isolation and functional characterization of a τ-cadinol synthase, a new sesquiterpene synthase from Lavandula angustifolia.

    Science.gov (United States)

    Jullien, Frédéric; Moja, Sandrine; Bony, Aurélie; Legrand, Sylvain; Petit, Cécile; Benabdelkader, Tarek; Poirot, Kévin; Fiorucci, Sébastien; Guitton, Yann; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis

    2014-01-01

    In this paper we characterize three sTPSs: a germacrene D (LaGERDS), a (E)-β-caryophyllene (LaCARS) and a τ-cadinol synthase (LaCADS). τ-cadinol synthase is reported here for the first time and its activity was studied in several biological models including transiently or stably transformed tobacco species. Three dimensional structure models of LaCADS and Ocimum basilicum γ-cadinene synthase were built by homology modeling using the template structure of Gossypium arboreum δ-cadinene synthase. The depiction of their active site organization provides evidence of the global influence of the enzymes on the formation of τ-cadinol: instead of a unique amino-acid, the electrostatic properties and solvent accessibility of the whole active site in LaCADS may explain the stabilization of the cadinyl cation intermediate. Quantitative PCR performed from leaves and inflorescences showed two patterns of expression. LaGERDS and LaCARS were mainly expressed during early stages of flower development and, at these stages, transcript levels paralleled the accumulation of the corresponding terpene products (germacrene D and (E)-β-caryophyllene). By contrast, the expression level of LaCADS was constant in leaves and flowers. Phylogenetic analysis provided informative results on potential duplication process leading to sTPS diversification in lavender.

  3. Evaluation of C-Reactive Protein Level in Patients with Pain Form of Temporomandibular Joint Dysfunction

    Directory of Open Access Journals (Sweden)

    Malgorzata Pihut

    2018-01-01

    Full Text Available Temporomandibular joint dysfunction is a functional disorder concerned with the abnormal functioning of the muscles of the stomatognathic system and temporomandibular joints involved in the dynamic movements of the jaw and surrounding structures. The aim of the study was to compare the level of C-reactive protein in patients with pain and painless forms of temporomandibular joint dysfunction. Materials and methods. The study group consisted of 72 patients who reported to the prosthetic treatment because of temporomandibular joint dysfunction. The study group included 36 patients with pain form of dysfunction, and the control group included 36 patients with painless form of disorder. Each patient underwent specialized examination of functional disorders in order to diagnose the type of dysfunction and was commissioned to carry out a study of the blood test concerned with evaluation of the C-reactive protein (CRP level in the same analytical laboratory. The results of the investigation were subjected to statistical analysis. The research obtained approval from the Ethics Committee of the Jagiellonian University (KBET/125/L/2013. Level of Evidence for primary research was established as type V. Results. The mean values of C-reactive protein levels in both groups were in the normal range and did not differ statistically significantly, which indicates the fact that the pain form of the temporomandibular joint disorders is not associated with inflammation of the soft tissues of the joint. Conclusion. Painful form of the temporomandibular joint dysfunctions is not connected with the inflammation of joints.

  4. Metformin induces oxidative stress in white adipocytes and raises uncoupling protein 2 levels.

    Science.gov (United States)

    Anedda, Andrea; Rial, Eduardo; González-Barroso, M Mar

    2008-10-01

    Metformin is a drug widely used to treat type 2 diabetes. It enhances insulin sensitivity by improving glucose utilization in tissues like liver or muscle. Metformin inhibits respiration, and the decrease in cellular energy activates the AMP-activated protein kinase that in turn switches on catabolic pathways. Moreover, metformin increases lipolysis and beta-oxidation in white adipose tissue, thereby reducing the triglyceride stores. The uncoupling proteins (UCPs) are transporters that lower the efficiency of mitochondrial oxidative phosphorylation. UCP2 is thought to protect against oxidative stress although, alternatively, it could play an energy dissipation role. The aim of this work was to analyse the involvement of UCP2 on the effects of metformin in white adipocytes. We studied the effect of this drug in differentiating 3T3-L1 adipocytes and found that metformin causes oxidative stress since it increases the levels of reactive oxygen species (ROS) and lowers the aconitase activity. Variations in UCP2 protein levels parallel those of ROS. Metformin also increases lipolysis in these cells although only when the levels of ROS and UCP2 have decreased. Hence, UCP2 does not appear to be needed to facilitate fatty acid oxidation. Furthermore, treatment of C57BL/6 mice with metformin also augmented the levels of UCP2 in epididymal white adipose tissue. We conclude that metformin treatment leads to the overexpression of UCP2 in adipocytes to minimize the oxidative stress that is probably due to the inhibition of respiration caused by the drug.

  5. Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

    Czech Academy of Sciences Publication Activity Database

    Benoni, Roberto; De Bei, O.; Paredi, G.; Hayes, C. S.; Franko, N.; Mozzarelli, A.; Bettati, S.; Campanini, B.

    2017-01-01

    Roč. 591, č. 9 (2017), s. 1212-1224 ISSN 0014-5793 Institutional support: RVO:61388963 Keywords : cysteine synthase * protein - protein interaction * serine acetyltransferase Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.623, year: 2016

  6. Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar.

    Science.gov (United States)

    Sousa, Daniele O B; Carvalho, Ana F U; Oliveira, José Tadeu A; Farias, Davi F; Castelar, Ivan; Oliveira, Henrique P; Vasconcelos, Ilka M

    2015-07-22

    The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed.

  7. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae.

    Science.gov (United States)

    Masser, Anna E; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan; Andréasson, Claes

    2016-05-01

    Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd.

  8. Infrared analyzers for breast milk analysis: fat levels can influence the accuracy of protein measurements.

    Science.gov (United States)

    Kwan, Celia; Fusch, Gerhard; Bahonjic, Aldin; Rochow, Niels; Fusch, Christoph

    2017-10-26

    Currently, there is a growing interest in lacto-engineering in the neonatal intensive care unit, using infrared milk analyzers to rapidly measure the macronutrient content in breast milk before processing and feeding it to preterm infants. However, there is an overlap in the spectral information of different macronutrients, so they can potentially impact the robustness of the measurement. In this study, we investigate whether the measurement of protein is dependent on the levels of fat present while using an infrared milk analyzer. Breast milk samples (n=25) were measured for fat and protein content before and after being completely defatted by centrifugation, using chemical reference methods and near-infrared milk analyzer (Unity SpectraStar) with two different calibration algorithms provided by the manufacturer (released 2009 and 2015). While the protein content remained unchanged, as measured by elemental analysis, measurements by infrared milk analyzer show a difference in protein measurements dependent on fat content; high fat content can lead to falsely high protein content. This difference is less pronounced when measured using the more recent calibration algorithm. Milk analyzer users must be cautious of their devices' measurements, especially if they are changing the matrix of breast milk using more advanced lacto-engineering.

  9. Effect of nonsurgical periodontal treatment on C-reactive protein levels in maintenance hemodialysis patients.

    Science.gov (United States)

    Yazdi, Farin Kiany; Karimi, Noozhan; Rasouli, Manoochehr; Roozbeh, Jamshid

    2013-01-01

    C-reactive protein (CRP) has been implicated as a possible mediator of the association between periodontitis and several systemic diseases. This study evaluated the impact of nonsurgical periodontal treatment on the serum levels of CRP in chronic kidney disease (CKD) patients on hemodialysis. A total of 77 CKD patients on hemodialysis were included in this study. At baseline, periodontal examination was assessed for all the patients, and chronic periodontitis was defined through clinical attachment level and probing pocket depth, according to the American Association of Periodontology. Nonsurgical periodontal treatment was performed and serum levels of CRP were evaluated at baseline and 8 weeks after periodontal treatment. Periodontal treatment resulted in significant reductions in CRP levels (p periodontitis. Periodontitis is an important source of systemic inflammation in CKD patients. Nonsurgical periodontal treatment can effectively reduce the serum level of CRP in these patients.

  10. Ultraviolet-ozone treatment reduces levels of disease-associated prion protein and prion infectivity

    Science.gov (United States)

    Johnson, C.J.; Gilbert, P.; McKenzie, D.; Pedersen, J.A.; Aiken, Judd M.

    2009-01-01

    Background. Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases caused by novel infectious agents referred to as prions. Prions appear to be composed primarily, if not exclusively, of a misfolded isoform of the cellular prion protein. TSE infectivity is remarkably stable and can resist many aggressive decontamination procedures, increasing human, livestock and wildlife exposure to TSEs. Findings. We tested the hypothesis that UV-ozone treatment reduces levels of the pathogenic prion protein and inactivates the infectious agent. We found that UV-ozone treatment decreased the carbon and prion protein content in infected brain homogenate to levels undetectable by dry-ashing carbon analysis or immunoblotting, respectively. After 8 weeks of ashing, UV-ozone treatment reduced the infectious titer of treated material by a factor of at least 105. A small amount of infectivity, however, persisted despite UV-ozone treatment. When bound to either montmorillonite clay or quartz surfaces, PrPTSE was still susceptible to degradation by UV-ozone. Conclusion. Our findings strongly suggest that UV-ozone treatment can degrade pathogenic prion protein and inactivate prions, even when the agent is associated with surfaces. Using larger UV-ozone doses or combining UV-ozone treatment with other decontaminant methods may allow the sterilization of TSE-contaminated materials. ?? 2009 Aiken et al; licensee BioMed Central Ltd.

  11. Intake of Mung Bean Protein Isolate Reduces Plasma Triglyceride Level in Rats

    Directory of Open Access Journals (Sweden)

    Nobuhiko Tachibana

    2013-09-01

    Full Text Available ABSTRACTBackground: Mung bean is well known as a starch source, but the physiological effects of mung bean protein have received little attention. In this study, we isolated mung bean protein from de-starched mung bean solutions, and investigated its influence on lipid metabolism. Objective: The aim of this study is to clarify the influence of the lipid metabolism by consumption of mung bean protein isolate (MPIMethods: Diets containing either mung bean protein isolate (MPI or casein were fed to normal rats for 28 days.Results: Both groups ate the same amount of food, but the plasma triglyceride level, relative liver weight and liver lipid contents (cholesterol and triglyceride pool in the MPI group were significantly lower than in the casein group. In the MPI group, the expression of sterol regulatory-element binding factor 1 (SREBF1 mRNA in the liver was significantly different when compared with the casein group. The significantly lower levels of insulin and free fatty acids in the MPI-fed rats may be due to the regulation of genes related to lipid metabolism in the liver.Conclusions: These results suggest that MPI may improve the plasma lipid profile by normalizing insulin sensitivity.Keywords: mung bean, Vigna radiata L., 8S globulin, triglyceride, β-conglycinin, 7S globulin, insulin sensitivity, SREBF1

  12. Producing biofuels using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  13. Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity

    DEFF Research Database (Denmark)

    Gojkovic, Zoran; Sandrini, Michael; Piskur, Jure

    2001-01-01

    no pyrimidine catabolic pathway, it enabled growth on N-carbamyl- beta -alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta -alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial......beta -Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamy-beta -alanine as the sole nitrogen source and exhibits diminished beta -alanine synthase...... N- carbamyl amidohydrolases. All three beta -alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N...

  14. Blood parameters in growing pigs fed increasing levels of bacterial protein meal

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, Anders

    2007-01-01

    The experiment investigated the effects of increasing dietary levels of bacterial protein meal (BPM) on various blood parameters reflecting protein and fat metabolism, liver function, and purine base metabolism in growing pigs. Sixteen barrows were allocated to four different experimental diets....... The control diet was based on soybean meal. In the other three diets soybean meal was replaced with increasing levels of BPM, approximately 17%, 35%, and 50% of the nitrogen being derived from BPM. Blood samples from the jugular vein were taken when the body weights of the pigs were approximately 10 kg, 21 kg......, 45 kg, and 77 kg. The blood parameters reflecting fat metabolism and liver funtion were not affected by diet. Both the plasma albumin and uric acid concentrations tended to decrease (P = 0.07 and 0.01, respectively) with increasing dietary BPM content, whereas the plasma glucose concentration tended...

  15. Serum levels of carbonylated and nitrosylated proteins in mobbing victims with workplace adjustment disorders.

    Science.gov (United States)

    Di Rosa, A E; Gangemi, S; Cristani, M; Fenga, C; Saitta, S; Abenavoli, E; Imbesi, S; Speciale, A; Minciullo, P L; Spatari, G; Abbate, S; Saija, A; Cimino, F

    2009-12-01

    Today the most important problem in the work place is psychological abuse, which may affect the health because of high levels of stress and anxiety. There is evidence that most psychiatric disorders are associated with increased oxidative stress but nothing is reported about the presence of oxidative stress in mobbing victims. This study has been carried out in a group of 19 patients affected by workplace mobbing-due adjustment disorders, in comparison with 38 healthy subjects, to evaluate whether oxidative stress may be induced by mobbing. Serum levels of protein carbonyl groups and of nitrosylated proteins, biological markers of oxidative stress conditions, were higher than those measured in healthy subjects. These findings may contribute to a better understanding of the redox homeostasis dysregulation occurring in victims of workplace mobbing.

  16. ATP Synthase, a Target for Dementia and Aging?

    Science.gov (United States)

    Larrick, James W; Larrick, Jasmine W; Mendelsohn, Andrew R

    2018-02-01

    Advancing age is the biggest risk factor for development for the major life-threatening diseases in industrialized nations accounting for >90% of deaths. Alzheimer's dementia (AD) is among the most devastating. Currently approved therapies fail to slow progression of the disease, providing only modest improvements in memory. Recently reported work describes mechanistic studies of J147, a promising therapeutic molecule previously shown to rescue the severe cognitive deficits exhibited by aged, transgenic AD mice. Apparently, J147 targets the mitochondrial alpha-F1-ATP synthase (ATP5A). Modest inhibition of the ATP synthase modulates intracellular calcium to activate AMP-activated protein kinase to inhibit mammalian target of rapamycin, a known mechanism of lifespan extension from worms to mammals.

  17. Factors Influencing the Measurement of Plasma/Serum Surfactant Protein D Levels by ELISA

    OpenAIRE

    Bratcher, Preston E.; Gaggar, Amit

    2014-01-01

    BACKGROUND: Extensive variations in human surfactant protein D (SP-D) levels in circulation as measured by ELISA exist in the published literature. In order to determine the source of these variations, factors influencing the measurement by ELISA were explored. MATERIALS AND METHODS: Peripheral blood from healthy individuals was collected into various vacutainers during the same blood draw. Recombinant SP-D was diluted into different matrices and used for a standard curve. Samples were analyz...

  18. Effect of physical training on glucose transporter protein and mRNA levels in rat adipocytes

    DEFF Research Database (Denmark)

    Stallknecht, B; Andersen, P H; Vinten, J

    1993-01-01

    Physical training increases insulin-stimulated glucose transport and the number of glucose transporters in adipocytes measured by cytochalasin B binding. In the present study we used immunoblotting to measure the abundance of two glucose transporters (GLUT-4, GLUT-1) in white adipocytes from....../or intrinsic activity). GLUT-1 protein and mRNA levels/adipocyte volume did not change with age or training....

  19. Supplementation of suckling beef calves with different levels of crude protein on tropical pasture.

    Science.gov (United States)

    Lopes, Sidnei Antonio; Paulino, Mário Fonseca; Detmann, Edenio; de Campos Valadares Filho, Sebastião; Valente, Eriton Egídio Lisboa; Barros, Lívia Vieira; Cardenas, Javier Enrique Garces; Almeida, Daniel Mageste; Martins, Leandro Soares; Silva, Aline Gomes

    2014-02-01

    The effects of supplementation with different levels of crude protein on performance, intake and nutrient digestibility and efficiency of microbial protein synthesis in suckling beef calves on pasture were assessed. Fifty-five calves, with an average age of 100 days and an initial average body weight of 110 ± 7.5 kg and their respective dams, were used. The experimental design was completely randomised with five treatments and 11 replications. The experimental treatments for calves were as follows: control = calves received only mineral mixture; supplementation levels = calves received supplement containing 8, 19, 30 or 41% of crude protein (CP, at a rate of 0.5% of body weight (BW)). The cows received only mineral mixture ad libitum. Supplemented calves had higher (P calves. There was no difference in total dry matter (DM) intake (P > 0.1). However, intake of dry matter forage (DMF) presented cubic profiles (P calves on creep feeding. The intake of supplements with CP levels between 8 and 30% partially replaces of the pasture ingested by calves and increases the digestibility of the diet.

  20. Effect of protein levels in rations on the immune response and body weight in fowl

    International Nuclear Information System (INIS)

    El-Abiad, N.M.F.

    1991-01-01

    The present study was carried out at animal experimental station at nuclear research centre. Enshas. Radioimmunological and biochemical assays were performed in laboratories of radioimmunology and biochemistry unite of atomic energy authority. It was planned investigate the effect of using different dietary protein levels on the antibody formation on female and male hubbard chicks. The study was also aimed to point out the effect of both dietary protein levels and thyroglobulin immunization on growth measurements and some metabolic blood parameters in relation to induced thyroid immunity to provide some insights on the autoimmune diseases in fowl. A total of 300 one - dau old Hubbard chicks (150 females and 150 males ) were used. Birds were reared on starter diet during the first two weeks of age then randomly classified into 3 groups of 50 of each sex; representing three nutritional diets applied up to the end of the experimental period that lasted 14 weeks. Low (14,44%), medium (18.41%) and high (22.11%) levels of protein in diet were fed to female and male chicks of the first, second and third group, respectively. All diets were formulated to be of equal energy and to cover the nutritional requirements recommended by NRC, (1984)

  1. Bifunctional effects of fucoidan on the expression of inducible nitric oxide synthase

    International Nuclear Information System (INIS)

    Yang, Jin Won; Yoon, Se Young; Oh, Soo Jin; Kim, Sang Kyum; Kang, Keon Wook

    2006-01-01

    Algal fucoidan is a marine sulfated polysaccharide with a wide variety of biological activities including anti-thrombotic and anti-inflammatory effects. This study evaluated the effect of fucoidan on the expression of inducible nitric oxide synthase (iNOS) in a macrophage cell line, RAW264.7. Low concentration range of fucoidan (10 μg/ml) increased the basal expression level of iNOS in quiescent macrophages. However, we found for the first time that fucoidan inhibited the release of nitric oxide (NO) in RAW264.7 cells stimulated with lipopolysaccharide (LPS). Western blot analysis revealed that fucoidan suppressed the LPS-induced expression of the inducible nitric oxide synthase (iNOS) gene. Moreover, the activation of both nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) are key steps in the transcriptional activation of the iNOS gene. Here, it was revealed that fucoidan selectively suppressed AP-1 activation, and that the activation of AP-1 appears to be essential for the induction of iNOS in activated macrophages. This inhibitory effect on AP-1 activation by fucoidan might be associated with its NO blocking and anti-inflammatory effects

  2. Chemotherapeutic treatment reduces circulating levels of surfactant protein-D in children with acute lymphoblastic leukemia

    DEFF Research Database (Denmark)

    Rathe, Mathias; Sorensen, Grith L; Skov Wehner, Peder

    2017-01-01

    with acute lymphoblastic leukemia (ALL). PROCEDURE: In a prospective study, 43 children receiving treatment for ALL were monitored for mucosal toxicity from diagnosis through the induction phase of treatment. Serial blood draws were taken to determine the levels of SP-D, interleukin-6 (IL-6), C......BACKGROUND: Surfactant protein D (SP-D) is a host defense molecule of the innate immune system that enhances pathogen clearance and modulates inflammatory responses. We hypothesized that circulating SP-D levels are associated with chemotherapy-induced mucositis and infectious morbidity in children...

  3. Serum levels of C-reactive protein in adolescents with periodontitis

    DEFF Research Database (Denmark)

    López, Rodrigo; Baelum, Vibeke; Hedegaard, Chris Juul

    2011-01-01

    Background: The results of several cross-sectional studies suggested a relationship between periodontitis and higher serum levels of C-reactive protein (CRP). Most of these studies were restricted to adult study groups with severe periodontal inflammation, and the potential effects of confounding...... ng/ml (31 to 183 ng/ml), respectively (P = 0.8). Conclusions: Serum levels of CRP were not significantly higher among subjects with periodontitis than among controls. However, a statistically significant positive association between percentages of sites with bleeding on probing and log...

  4. Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cell Suspensions: A Proteomic Analysis.

    Science.gov (United States)

    Gas-Pascual, Elisabet; Simonovik, Biljana; Heintz, Dimitri; Bergdoll, Marc; Schaller, Hubert; Bach, Thomas J

    2015-08-01

    The effect of an inhibitor of cycloartenol synthase (CAS, EC 5.4.99.8) on the proteome of tobacco BY-2 cells has been examined. CAS catalyzes the first committed step in phytosterol synthesis in plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Proteins were separated by two-dimensional electrophoresis and spots, that clearly looked differentially accumulated after visual inspection, were cut, in-gel trypsin digested, and peptides were analyzed by nano-HPLC-MS/MS. Distinct peptides were compared to sequences in the data banks and attributed to corresponding proteins and genes. Inhibition of CAS induced proteins that appear to mitigate the negative effects of the chemical exposure. However, as all enzymes that are directly involved in phytosterol biosynthesis are low-abundant proteins, significant changes in their levels could not be observed. Differences could be seen with enzymes involved in primary metabolism (glycolysis, pentose phosphate pathway etc.), in proteins of the chaperonin family, and those, like actin, that participate in formation and strengthening of the cytoskeleton and have some impact on cell growth and division.

  5. Regulation of glycogen synthase kinase-3 during bipolar mania treatment.

    Science.gov (United States)

    Li, Xiaohong; Liu, Min; Cai, Zhuoji; Wang, Gang; Li, Xiaohua

    2010-11-01

    Bipolar disorder is a debilitating psychiatric illness presenting with recurrent mania and depression. The pathophysiology of bipolar disorder is poorly understood, and molecular targets in the treatment of bipolar disorder remain to be identified. Preclinical studies have suggested that glycogen synthase kinase-3 (GSK3) is a potential therapeutic target in bipolar disorder, but evidence of abnormal GSK3 in human bipolar disorder and its response to treatment is still lacking. This study was conducted in acutely ill type I bipolar disorder subjects who were hospitalized for a manic episode. The protein level and the inhibitory serine phosphorylation of GSK3 in peripheral blood mononuclear cells of bipolar manic and healthy control subjects were compared, and the response of GSK3 to antimanic treatment was evaluated. The levels of GSK3α and GSK3β in this group of bipolar manic subjects were higher than healthy controls. Symptom improvement during an eight-week antimanic treatment with lithium, valproate, and atypical antipsychotics was accompanied by a significant increase in the inhibitory serine phosphorylation of GSK3, but not the total level of GSK3, whereas concomitant electroconvulsive therapy treatment during a manic episode appeared to dampen the response of GSK3 to pharmacological treatment. Results of this study suggest that GSK3 can be modified during the treatment of bipolar mania. This finding in human bipolar disorder is in agreement with preclinical data suggesting that inhibition of GSK3 by increasing serine phosphorylation is a response of GSK3 to psychotropics used in bipolar disorder, supporting the notion that GSK3 is a promising molecular target in the pharmacological treatment of bipolar disorder. © 2010 John Wiley and Sons A/S.

  6. Isolation and characterization of an oxidosqualene cyclase gene encoding a β-amyrin synthase involved in Polygala tenuifolia Willd. saponin biosynthesis.

    Science.gov (United States)

    Jin, Mei Lan; Lee, Dae Young; Um, Yurry; Lee, Jeong Hoon; Park, Chun Geun; Jetter, Reinhard; Kim, Ok Tae

    2014-03-01

    Expression of PtBS (Polygala tenuifolia β-amyrin synthase) led to the production of β-amyrin as sole product. Polygala tenuifolia Willdenow is a rich source of triterpene saponins, onjisaponins and polygalasaponins, used as herbal medicine to treat phlegms and for detumescence in traditional Asian healing. The Polygala saponins share the oleanane backbone structure and are, therefore, likely synthesized via β-amyrin as a common precursor. We hypothesized that, in analogy to diverse other plant species, this central intermediate should be formed by a β-amyrin synthase catalyzing the complex cyclization of oxidosqualene. This member of the oxidosqualene cyclase (OSC) family of enzymes is thus defining an important branch point between primary and secondary metabolisms, and playing a crucial role in the control of oleanane-type triterpene saponin biosynthesis. From P. tenuifolia roots, we isolated an OSC cDNA containing a reading frame of 2,289 bp nucleotides. The predicted protein of 763 amino acids (molecular weight 87.353 kDa) showed particularly high amino acid sequence identities to known β-amyrin synthases (85-87 %) and was, therefore, named PtBS. Expression of PtBS in the triterpenoid synthase-deficient yeast mutant GIL77 led to the production of β-amyrin as sole product. qRT-PCR analysis of various P. tenuifolia organs showed that PtBS transcript levels were highest in the roots, consistent with onjisaponin accumulation patterns. Therefore, we conclude that PtBS is the β-amyrin synthase enzyme catalyzing the first committed step in the biosynthesis of onjisaponins and polygalasaponins in P. tenuifolia.

  7. Effects of Dietary Protein and Lipid Levels on Growth and Body Composition of Juvenile Far Eastern Catfish

    Directory of Open Access Journals (Sweden)

    Kyoung-Duck Kim

    2012-03-01

    Full Text Available A 3×2 factorial experiment was conducted to determine the effects of dietary protein and lipid levels on the growth and body composition of juvenile far eastern catfish. Six diets were formulated to contain three levels of protein (20%, 30% and 40% and two levels of lipid (9% and 17%. Triplicate groups of fish (initial body weight of 7.6 g were hand-fed to apparent satiation for 66 days. Final mean weight was improved with increasing dietary protein and lipid levels, and the highest final mean weight was observed in fish fed the 40/17 (% protein/% lipid diet. No significant difference was observed in final mean weight for fish fed between 30/17 diet and 40/9 diet. Feed efficiency of fish fed the diets containing over 30% protein levels with 9% and 17% lipid levels were significantly higher than those of fish fed the 20% protein levels. Feed efficiency of fish fed the 30/17 diet was not significantly different from that of fish fed the 40/9 diet or 40/17 diet. Feed efficiency and protein efficiency ratio of fish fed the 20% protein diets with 17% lipid level were significantly higher than those of fish fed 9% lipid diet. Daily feed intake of fish tended to decrease with increasing dietary protein and lipid levels. Moisture content of whole body in fish fed the 9% lipid diets was significantly higher than that of fish fed the 17% lipid diets at the same protein level, but the opposite trends were found for crude lipid content. Significant effects of dietary lipid were observed for most fatty acids, according to their relative values in the diets. The results of this study suggest that the protein requirement for maximum growth of juvenile far eastern catfish may be higher than 40%, and an increase of dietary lipid level from 9% to 17% can improve growth and feed utilization.

  8. Identification of genes encoding granule-bound starch synthase involved in amylose metabolism in banana fruit.

    Directory of Open Access Journals (Sweden)

    Hongxia Miao

    Full Text Available Granule-bound starch synthase (GBSS is responsible for amylose synthesis, but the role of GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage.

  9. Identification of Genes Encoding Granule-Bound Starch Synthase Involved in Amylose Metabolism in Banana Fruit

    Science.gov (United States)

    Liu, Weixin; Xu, Biyu; Jin, Zhiqiang

    2014-01-01

    Granule-bound starch synthase (GBSS) is responsible for amylose synthesis, but the role of GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage. PMID:24505384

  10. Serum heart type fatty acid binding protein levels are not changed in hyperthyroidism.

    Science.gov (United States)

    Ozbek, Mustafa; Gungunes, Askin; Sahin, Mustafa; Ginis, Zeynep; Ucan, Bekir; Sayki, Muyesser; Tutal, Esra; Cakal, Erman; Kuşkonmaz, Serife M; Öztürk, Mehmet A; Delibasi, Tuncay

    2016-09-01

    Heart type fatty acid binding protein (H-FABP) is a small protein and released into the circulation when myocardial damage has occurred. Previous studies have demonstrated that H-FABP is closely associated with cardiac and some endocrinologic disorders including prediabetes, metabolic syndrome, and acromegaly. Hyperthyroism is a well-known disorder associated with cardiovascular diseases. We aimed to investigate the effect of hyperthyrodism on H-FABP levels. Forty six patients with hyperthyroidism with no known history of coronary artery disease and 40 healthy controls are involved in the study. Serum H-FABP levels are measured using sandwich enzyme-linked immunosorbent assay. There was no significant difference between serum H-FABP levels of patients with hyperthyroidism and controls (871±66 pg/mL, and 816±66 pg/mL, respectively P=0.56). There was no significant correlation between H-FABP, free triiodothyronine (fT3), free thyroxine (fT4), and thyroid stimulating hormone (TSH) levels in patients and controls. Serum H-FABP levels are not altered in patients with hyperthyroidism.

  11. Occupational exposure levels of bioaerosol components are associated with serum levels of the acute phase protein Serum Amyloid A in greenhouse workers

    DEFF Research Database (Denmark)

    Madsen, Anne Mette; Thilsing, Trine; Bælum, Jesper

    2016-01-01

    to elevated levels of bioaerosols. The objective of this study is to assess whether greenhouse workers personal exposure to bioaerosol components was associated with serum levels of the acute phase proteins Serum Amyloid A (SAA) and C-reactive protein (CRP). METHODS: SAA and CRP levels were determined......-glucan. RESULTS: Serum levels of SAA and CRP were not significantly different in greenhouse workers and a reference group, or on the two work days. In a mixed model, SAA levels were positively associated with endotoxin exposure levels (p = 0.0007). Results for fungi were not clear. CRP levels were positively...... associated with endotoxin exposures (p = 0.022). Furthermore, when workers were categorized into three groups based on SAA and CRP serum levels endotoxin exposure was highest in the group with the highest SAA levels and in the group with middle and highest CRP levels. SAA and CRP levels were elevated...

  12. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  13. Evaluation of the C-reactive protein serum levels in periodontitis patients with or without atherosclerosis.

    Science.gov (United States)

    Thakare, Kaustubh S; Deo, Vikas; Bhongade, Manohar L

    2010-01-01

    Several studies suggested an association between periodontal disease and cardiovascular disease (CVD). C- reactive protein is elevated in periodontitis patients and has been found to be a predictor of increased risk for cardiovascular disease. Since, CRP is known to play a role in pathogenesis of atherosclerosis, the present study was undertaken to evaluate the serum levels of CRP in periodontitis patients with or without atherosclerosis. A total of 45 patients, 15 chronic periodontitis patients with atherosclerosis (Group A), 15 chronic periodontitis patients with no history of any systemic disease (Group B), and 15 clinically healthy individuals with no history of periodontal or systemic disease (Group C) within age range of 30 to 55 years were selected for the study. PI, PBI, PPD, CAL and radiographic marginal alveolar bone level were assessed in all the three groups. CRP levels were assessed with 'Turbi-latex' kit using turbidimetric analysis. The mean CAL recorded was 4.9mm in group A, 4.6mm in group B and 1.9 mm in group C. The mean radiographic marginal bone level was 45 to 50% in group A, 45 to 50% in group B and 90 to 95% in group C. Mean serum C-reactive protein level was significantly higher in group A (8.9 mg/l), as compared to group B (4.9 mg/l) as well as group C (0.9 mg/l). Within the limits of this study it was concluded that periodontitis may add to the inflammatory burden of the individual and may result in increased risk of atherosclerosis based on serum C-reactive protein concentrations.

  14. Maternal high-fat diet and offspring expression levels of vitamin K-dependent proteins.

    Science.gov (United States)

    Lanham, S A; Cagampang, F R; Oreffo, R O C

    2014-12-01

    Studies suggest that bone growth and development and susceptibility to vascular disease in later life are influenced by maternal nutrition during intrauterine and early postnatal life. There is evidence for a role of vitamin K-dependent proteins (VKDPs) including osteocalcin, matrix Gla protein, periostin, and growth-arrest specific- protein 6, in both bone and vascular development. We have examined whether there are alterations in these VKDPs in bone and vascular tissue from offspring of mothers subjected to a nutritional challenge: a high-fat diet during pregnancy and postnatally, using 6-week-old mouse offspring. Bone site-specific and sex-specific differences across femoral and vertebral bone in male and female offspring were observed. Overall a high-fat maternal diet and offspring diet exacerbated the bone changes observed. Sex-specific differences and tissue-specific differences were observed in VKDP levels in aorta tissue from high-fat diet-fed female offspring from high-fat diet-fed mothers displaying increased levels of Gas6 and Ggcx compared with those of female controls. In contrast, differences were seen in VKDP levels in femoral bone of female offspring with lower expression levels of Mgp in offspring of mothers fed a high-fat diet compared with those of controls. We observed a significant correlation in Mgp expression levels within the femur to measures of bone structure of the femur and vertebra, particularly in the male offspring cohort. In summary, the current study has highlighted the importance of maternal nutrition on offspring bone development and the correlation of VKDPs to bone structure.

  15. Iron Loading Selectively Increases Hippocampal Levels of Ubiquitinated Proteins and Impairs Hippocampus-Dependent Memory.

    Science.gov (United States)

    Figueiredo, Luciana Silva; de Freitas, Betânia Souza; Garcia, Vanessa Athaíde; Dargél, Vinícius Ayub; Köbe, Luiza Machado; Kist, Luiza Wilges; Bogo, Maurício Reis; Schröder, Nadja

    2016-11-01

    Alterations of brain iron levels have been observed in a number of neurodegenerative disorders. We have previously demonstrated that iron overload in the neonatal period results in severe and persistent memory deficits in the adulthood. Protein degradation mediated by the ubiquitin-proteasome system (UPS) plays a central regulatory role in several cellular processes. Impairment of the UPS has been implicated in the pathogenesis of neurodegenerative disorders. Here, we examined the effects of iron exposure in the neonatal period (12th-14th day of postnatal life) on the expression of proteasome β-1, β-2, and β-5 subunits, and ubiquitinated proteins in brains of 15-day-old rats, to evaluate the immediate effect of the treatment, and in adulthood to assess long-lasting effects. Two different memory types, emotionally motivated conditioning and object recognition were assessed in adult animals. We found that iron administered in the neonatal period impairs both emotionally motivated and recognition memory. Polyubiquitinated protein levels were increased in the hippocampus, but not in the cortex, of adult animals treated with iron. Gene expression of subunits β1 and β5 was affected by age, being higher in the early stages of development in the hippocampus, accompanied by an age-related increase in polyubiquitinated protein levels in adults. In the cortex, gene expression of the three proteasome subunits was significantly higher in adulthood than in the neonatal period. These findings suggest that expression of proteasome subunits and activity are age-dependently regulated. Iron exposure in the neonatal period produces long-lasting harmful effects on the UPS functioning, which may be related with iron-induced memory impairment.

  16. Mapping Hydrophobicity on the Protein Molecular Surface at Atom-Level Resolution

    Science.gov (United States)

    Nicolau Jr., Dan V.; Paszek, Ewa; Fulga, Florin; Nicolau, Dan V.

    2014-01-01

    A precise representation of the spatial distribution of hydrophobicity, hydrophilicity and charges on the molecular surface of proteins is critical for the understanding of the interaction with small molecules and larger systems. The representation of hydrophobicity is rarely done at atom-level, as this property is generally assigned to residues. A new methodology for the derivation of atomic hydrophobicity from any amino acid-based hydrophobicity scale was used to derive 8 sets of atomic hydrophobicities, one of which was used to generate the molecular surfaces for 35 proteins with convex structures, 5 of which, i.e., lysozyme, ribonuclease, hemoglobin, albumin and IgG, have been analyzed in more detail. Sets of the molecular surfaces of the model proteins have been constructed using spherical probes with increasingly large radii, from 1.4 to 20 Å, followed by the quantification of (i) the surface hydrophobicity; (ii) their respective molecular surface areas, i.e., total, hydrophilic and hydrophobic area; and (iii) their relative densities, i.e., divided by the total molecular area; or specific densities, i.e., divided by property-specific area. Compared with the amino acid-based formalism, the atom-level description reveals molecular surfaces which (i) present an approximately two times more hydrophilic areas; with (ii) less extended, but between 2 to 5 times more intense hydrophilic patches; and (iii) 3 to 20 times more extended hydrophobic areas. The hydrophobic areas are also approximately 2 times more hydrophobicity-intense. This, more pronounced “leopard skin”-like, design of the protein molecular surface has been confirmed by comparing the results for a restricted set of homologous proteins, i.e., hemoglobins diverging by only one residue (Trp37). These results suggest that the representation of hydrophobicity on the protein molecular surfaces at atom-level resolution, coupled with the probing of the molecular surface at different geometric resolutions

  17. Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

    Energy Technology Data Exchange (ETDEWEB)

    Kanginakudru, Sriramana, E-mail: skangina@iu.edu [Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN (United States); DeSmet, Marsha, E-mail: mdesmet@iupui.edu [Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN (United States); Thomas, Yanique, E-mail: ysthomas@umail.iu.edu [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN (United States); Morgan, Iain M., E-mail: immorgan@vcu.edu [VCU Philips Institute for Oral Health Research, Virginia Commonwealth University, Richmond, Virginia (United States); Androphy, Elliot J., E-mail: eandro@iu.edu [Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN (United States); Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN (United States)

    2015-04-15

    The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication.

  18. Lung surfactant levels are regulated by Ig-Hepta/GPR116 by monitoring surfactant protein D.

    Directory of Open Access Journals (Sweden)

    Taku Fukuzawa

    Full Text Available Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta(+/+ and Ig-Hepta(-/- mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i balanced synthesis of surfactant lipids and proteins and (ii surfactant secretion, and (iii a stimulating effect on recycling (uptake in response to elevated levels of Sp-D in alveolar space.

  19. Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

    International Nuclear Information System (INIS)

    Kanginakudru, Sriramana; DeSmet, Marsha; Thomas, Yanique; Morgan, Iain M.; Androphy, Elliot J.

    2015-01-01

    The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication

  20. Effect of dietary protein level on ewe milk yield and on air quality under different ventilation rates

    Directory of Open Access Journals (Sweden)

    A. Sevi

    2010-01-01

    Full Text Available The efficiency of dietary N utilization for milk protein synthesis in dairy animals is quite low (15 to 35% (NRC, 1988; Tamminga, 1992, therefore farmers are driven to use high protein level diets for sustaining milk production in lactating animals. Previous experiments have demonstrated that an increase in the protein level of diet from 13 to 16% resulted in higher blood urea concentrations (Jaime and Purroy, 1995 and increased N excretion in urine in sheep (Gonzalez et al., 1984.

  1. Study of p53 protein expression levels from irradiated peripheral blood lymphocytes for biodosimetry

    International Nuclear Information System (INIS)

    Cavalcanti, M.B.; Fernandes, T.S.; Melo, J.A.; Neves, M.A.B.; Machado, C.G.F

    2005-01-01

    Biodosimetry can be defined as the investigation of radioinduced biological effects in order to correlate them with the absorbed dose. Scoring of unstable chromosomal aberrations and micronuclei, from in vitro irradiated peripheral blood lymphocytes, is commonly used for biodosimetry based on cytogenetic analysis. However, this method of analysis is time-consuming, which may represent a pitfall when fast investigation of a possible exposure to ionizing radiation (IR) is needed. The interaction of IR with the living cell can cause injuries in the DNA molecules. However, normal cells possess mechanisms of repair that are capable to correct those damages. During the repair process of the DNA various proteins are expressed. Among these proteins, p53 plays an important role. This protein is a transcription factor that helps in the maintenance of the genomic integrity. p53 protein is found into the cytoplasm in reduced concentrations and has a short average life. However, expression of p53 protein can be induced by DNA harmful radioinduced, which increases the concentration and the average life of this protein, making possible its detection. Thus, the correlation between the increasing of p53 expression and the irradiation may constitute a fast and reliable method of individual monitoring in cases of accidental or suspected exposures to IR. In this context, the objective of this research was to evaluate the p53 protein expression levels from lymphocytes of the human peripheral blood after in vitro irradiation. For this, samples of peripheral blood from healthy individuals were irradiated with known doses. Lymphocytes were separated on ficoll gradient by centrifugation and re-suspended at 1x 10 6 /mL in RPMI medium enriched with fetal calf serum. Hence, lymphocytes were incubated in 5% CO 2 at 37 deg C prior to the methodology of flow cytometry, using intranuclear antigens for the quantification of p53. In this report, the methodology performed and the results obtained

  2. Study of p53 protein expression levels from irradiated peripheral blood lymphocytes for biodosimetry

    Energy Technology Data Exchange (ETDEWEB)

    Cavalcanti, M.B.; Fernandes, T.S. [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Energia Nuclear; Amaral, A. [Universite Paris XII (UPXII) (France); Melo, J.A. [Centro de Radioterapia de Pernambuco (CERAPE), PE (Brazil); Neves, M.A.B.; Machado, C.G.F, E-mail: maribrayner@yahoo.com.br [Fundacao de Hematologia e Hemoterapia de Pernambuco, PE (Brazil)

    2005-07-01

    Biodosimetry can be defined as the investigation of radioinduced biological effects in order to correlate them with the absorbed dose. Scoring of unstable chromosomal aberrations and micronuclei, from in vitro irradiated peripheral blood lymphocytes, is commonly used for biodosimetry based on cytogenetic analysis. However, this method of analysis is time-consuming, which may represent a pitfall when fast investigation of a possible exposure to ionizing radiation (IR) is needed. The interaction of IR with the living cell can cause injuries in the DNA molecules. However, normal cells possess mechanisms of repair that are capable to correct those damages. During the repair process of the DNA various proteins are expressed. Among these proteins, p53 plays an important role. This protein is a transcription factor that helps in the maintenance of the genomic integrity. p53 protein is found into the cytoplasm in reduced concentrations and has a short average life. However, expression of p53 protein can be induced by DNA harmful radioinduced, which increases the concentration and the average life of this protein, making possible its detection. Thus, the correlation between the increasing of p53 expression and the irradiation may constitute a fast and reliable method of individual monitoring in cases of accidental or suspected exposures to IR. In this context, the objective of this research was to evaluate the p53 protein expression levels from lymphocytes of the human peripheral blood after in vitro irradiation. For this, samples of peripheral blood from healthy individuals were irradiated with known doses. Lymphocytes were separated on ficoll gradient by centrifugation and re-suspended at 1x 10{sub 6}/mL in RPMI medium enriched with fetal calf serum. Hence, lymphocytes were incubated in 5% CO{sub 2} at 37 deg C prior to the methodology of flow cytometry, using intranuclear antigens for the quantification of p53. In this report, the methodology performed and the results

  3. Development of multigene expression signature maps at the protein level from digitized immunohistochemistry slides.

    Directory of Open Access Journals (Sweden)

    Gregory J Metzger

    Full Text Available Molecular classification of diseases based on multigene expression signatures is increasingly used for diagnosis, prognosis, and prediction of response to therapy. Immunohistochemistry (IHC is an optimal method for validating expression signatures obtained using high-throughput genomics techniques since IHC allows a pathologist to examine gene expression at the protein level within the context of histologically interpretable tissue sections. Additionally, validated IHC assays may be readily implemented as clinical tests since IHC is performed on routinely processed clinical tissue samples. However, methods have not been available for automated n-gene expression profiling at the protein level using IHC data. We have developed methods to compute expression level maps (signature maps of multiple genes from IHC data digitized on a commercial whole slide imaging system. Areas of cancer for these expression level maps are defined by a pathologist on adjacent, co-registered H&E slides, allowing assessment of IHC statistics and heterogeneity within the diseased tissue. This novel way of representing multiple IHC assays as signature maps will allow the development of n-gene expression profiling databases in three dimensions throughout virtual whole organ reconstructions.

  4. Selective effects of whey protein concentrate on glutathione levels and apoptosis in rats with mammary tumors.

    Science.gov (United States)

    Cheng, Shih-Hsuan; Tseng, Yang-Ming; Wu, Szu-Hsien; Tsai, Shih-Meng; Tsai, Li-Yu

    2017-09-01

    Glutathione (GSH) plays an important role in antioxidant defense and regulation of apoptosis. GSH deficiency is related to many diseases, including cancer, and increased GSH levels in cancer cells are associated with chemotherapy resistance because of resistance to apoptosis. In this study, we investigated the effects of whey protein concentrate (WPC), a precursor of GSH, in rats with mammary tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (DMBA). DMBA treatment results in cellular changes that mimic the initiation and promotion of carcinogenesis of breast tissue. We aimed to examine the possible preventive effects of diets containing whey protein on DMBA-induced mammary tumors in rats. The results indicate that WPC (0.334 g/kg) supplementation significantly increased the liver GSH levels by 92%, and were accompanied by low Bax/Bcl-2 ratio (from 5 to 3) and cleaved caspase-3/procaspase-3 ratio (from 2.4 to 1.2) in DMBA-treated rats. Furthermore, tumor GSH levels were decreased by 47% in WPC-supplemented rats, which resulted in increased Bax/Bcl-2 ratio (from 0.9 to 2) and cleaved caspase-3/procaspase-3 ratio (from 1.1 to 2.7). In conclusion, supplementation with WPC could selectively deplete tumor GSH levels and, therefore, WPC supplementation might be a promising strategy to overcome treatment resistance in cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Effect of Dietary Protein Levels on Composition of Odorous Compounds and Bacterial Ecology in Pig Manure

    Directory of Open Access Journals (Sweden)

    Sungback Cho

    2015-09-01

    Full Text Available This study was performed to investigate the effect of different levels of dietary crude protein (CP on composition of odorous compounds and bacterial communities in pig manure. A total of 48 male pigs (average initial body weight 45 kg fed diets containing three levels of dietary CP (20%, 17.5%, and 15% and their slurry samples were collected from the pits under the floor every week for one month. Changes in composition of odorous compounds and bacterial communities were analyzed by gas chromatography and 454 FLX titanium pyrosequencing systems, respectively. Levels of phenols, indoles, short chain fatty acid and branched chain fatty acid were lowest (p<0.05 in CP 15% group among three CP levels. Relative abundance of Bacteroidetes phylum and bacterial genera including Leuconostoc, Bacillus, Atopostipes, Peptonphilus, Ruminococcaceae_uc, Bacteroides, and Pseudomonas was lower (p<0.05 in CP 15% than in CP 20% group. There was a positive correlation (p<0.05 between odorous compounds and bacterial genera: phenol, indole, iso-butyric acid, and iso-valeric acid with Atopostipes, p-cresol and skatole with Bacteroides, acetic acid and butyric acid with AM982595_g of Porphyromonadaceae family, and propionic acid with Tissierella. Taken together, administration of 15% CP showed less production of odorous compounds than 20% CP group and this result might be associated with the changes in bacterial communities especially whose roles in protein metabolism.

  6. Plasma lactate, GH and GH-binding protein levels in exercise following BCAA supplementation in athletes.

    Science.gov (United States)

    De Palo, E F; Gatti, R; Cappellin, E; Schiraldi, C; De Palo, C B; Spinella, P

    2001-01-01

    Branched chain amino acids (BCAA) stimulate protein synthesis, and growth hormone (GH) is a mediator in this process. A pre-exercise BCAA ingestion increases muscle BCAA uptake and use. Therefore after one month of chronic BCAA treatment (0.2 gkg(-1) of body weight), the effects of a pre-exercise oral supplementation of BCAA (9.64 g) on the plasma lactate (La) were examined in triathletes, before and after 60 min of physical exercise (75% of VO2 max). The plasma levels of GH (pGH) and of growth hormone binding protein (pGHBP) were also studied. The end-exercise La of each athlete was higher than basal. Furthermore, after the chronic BCAA treatment, these end-exercise levels were lower than before this treatment (8.6+/-0.8 mmol L(-1) after vs 12.8+/-1.0 mmol L(-1) before treatment; p BCAA chronic treatment, this end-exercise pGHBP was 738+/-85 pmol L(-1) before vs 1691+/-555 pmol L(-1) after. pGH/pGHBP ratio was unchanged in each athlete and between the groups, but a tendency to increase was observed at end-exercise. The lower La at the end of an intense muscular exercise may reflect an improvement of BCAA use, due to the BCAA chronic treatment. The chronic BCAA effects on pGH and pGHBP might suggest an improvement of muscle activity through protein synthesis.

  7. A cytosolic copper storage protein provides a second level of copper tolerance in Streptomyces lividans.

    Science.gov (United States)

    Straw, Megan L; Chaplin, Amanda K; Hough, Michael A; Paps, Jordi; Bavro, Vassiliy N; Wilson, Michael T; Vijgenboom, Erik; Worrall, Jonathan A R

    2018-01-24

    Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P 1 -type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Δccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance.

  8. Ischemia - reperfusion induced changes in levels of ion transport proteins in gerbil brain

    International Nuclear Information System (INIS)

    Lehotsky, J.; Racay, P.; Kaplan, P.; Mezesova, V.; Raeymaekers, L.

    1998-01-01

    A quantitative Western blotting was used to asses the levels of ion transport proteins in gerbil brain in control and in animals after ischemic-reperfusion injury (IRI). The gene products of plasma membrane Ca 2+ pump (PMCA) were detected in the hippocampus, cerebral cortex and cerebellum. However, they showed a distinct distribution pattern. Inositol 1,4,5-triphosphate (Ins 3 ) receptor and reticular Ca 2+ pump are the most abundant in cerebellum and hippocampus. The IRI leads to a selective decrease in content of PMCA and InsP 3 receptor I isoforms. The levels of α 3 isoform of Na + pump and reticular proteins: Ca 2+ pump and calreticulin remained constant. InsP 3 receptor and organellar Ca 2+ (SERCA) are the most abundant in cerebellum and hippocampus. Ischemia and reperfusion up to 10 days leads to a signal decrease of PMCA immuno-signal. We suppose that alteration of number of ion transport proteins, can contribute to changes which participate or follow the delayed death of neurons in hippocampus. (authors)

  9. Gestational Protein Restriction Increases Cardiac Connexin 43 mRNA levels in male adult rat offspring

    Science.gov (United States)

    Rossini, Kamila Fernanda; de Oliveira, Camila Andrea; Rebelato, Hércules Jonas; Esquisatto, Marcelo Augusto Marreto; Catisti, Rosana

    2017-01-01

    Background The dietary limitation during pregnancy influences the growth and development of the fetus and offspring and their health into adult life. The mechanisms underlying the adverse effects of gestational protein restriction (GPR) in the development of the offspring hearts are not well understood. Objectives The aim of this study was to evaluate the effects of GPR on cardiac structure in male rat offspring at day 60 after birth (d60). Methods Pregnant Wistar rats were fed a normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. Blood pressure (BP) values from 60-day-old male offspring were measured by an indirect tail-cuff method using an electro sphygmomanometer. Hearts (d60) were collected for assessment of connexin 43 (Cx43) mRNA expression and morphological and morphometric analysis. Results LP offspring showed no difference in body weight, although they were born lighter than NP offspring. BP levels were significantly higher in the LP group. We observed a significant increase in the area occupied by collagen fibers, a decrease in the number of cardiomyocytes by 104 µm2, and an increase in cardiomyocyte area associated with an increased Cx43 expression. Conclusion GPR changes myocardial levels of Cx43 mRNA in male young adult rats, suggesting that this mechanism aims to compensate the fibrotic process by the accumulation of collagen fibers in the heart interstitium. PMID:28678925

  10. Effect of changes in dietary protein and oil levels on production parameters of female broiler chickens

    International Nuclear Information System (INIS)

    Farran, M.T.; Barbour, G.W.; Usayran, N.N.; Darwish, A.

    2013-01-01

    Two experiments, as factorial arrangement of treatments in a complete randomized design, were conducted to evaluate weight gain (WG), feed conversion (FC), and carcass characteristics of female broilers fed diets varying in crude protein (CP) and metabolisable energy (ME) levels with graded oil supplementation. In experiment 1, the CP level was 190 and 220 g/kg in the starter diets and reduced by 25 g/kg for each grower diet with ME of 12.1 and 12.6 MJ /kg and oil level of 0 and 40 g/kg. In the second experiment, the level of CP was 190, 210, and 230 g/kg in the starter diets and reduced by 30g/kg in each corresponding grower diet with an oil level of 0, 20, and 40 g /kg. The 190 g/kg dietary CP reduced WG of birds at market age in both experiments but increased the FC value only in trial 2 (P < 0.05). In addition, it reduced protein and moisture contents but increased fat level in ready to cook (RTC) carcasses (P<0.05). In experiment 2, however, birds fed the 210 g CP/kg diet had WG and FC at market age, and yield of abdominal fat, pectoralis major muscle and drum, in addition to RTC carcass moisture comparable to those fed the highest dietary CP level. Dietary oil supplementation at 40 g/kg improved (P<0.05) bird WG and FC in both trials. In conclusion, diets containing 40 g oil/kg with 210 - 180 g CP/kg (starter and grower, respectively) can be safely fed to broiler females. (author)

  11. Seasonal influence on gene expression of monoterpene synthases in Salvia officinalis (Lamiaceae).

    Science.gov (United States)

    Grausgruber-Gröger, Sabine; Schmiderer, Corinna; Steinborn, Ralf; Novak, Johannes

    2012-03-01

    Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and β-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/β-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively). Copyright © 2011 Elsevier GmbH. All rights reserved.

  12. Renal and urinary levels of endothelial protein C receptor correlate with acute renal allograft rejection.

    Directory of Open Access Journals (Sweden)

    Lionel Lattenist

    Full Text Available The Endothelial Protein C Receptor (EPCR is expressed on leukocytes, on endothelium of large blood vessels and to a lesser extent on capillaries. Membrane bound EPCR plays an important role in the activation of protein C which has anticoagulant, anti-inflammatory and cytoprotective effects. After cleavage by a protease EPCR is also found as a soluble protein. Acute rejection of kidney allografts can be divided in T-cell-mediated rejection (TCMR and antibody-mediated (ABMR rejection. The latter is characterized by strong activation of coagulation. Currently no reliable non-invasive biomarkers are available to monitor rejection. Renal biopsies were available from 81 renal transplant patients (33 without rejection, 26 TCMR and 22 ABMR, we had access to mRNA material, matched plasma and urine samples for a portion of this cohort. Renal EPCR expression was assessed by RT-PCR and immunostaining. Plasma and urine sEPCR levels were measured by ELISA. ABMR patients showed higher levels of EPCR mRNA than TCMR patients. EPCR expression on glomeruli was significantly elevated in ABMR patients than in TCMR or control patients. In the peritubular capillaries EPCR expression was higher in ABMR patients than in control patients. EPCR expression was higher in tubules and arteries of rejection patients than in control patients. Plasma sEPCR levels did not differ. Urine sEPCR levels were more elevated in the ABMR group than in patients with TCMR or without rejection. ROC analysis demonstrated that urinary sEPCR is appropriate to discriminate between ABMR patients and TCMR or control patients. We conclude that urinary sEPCR could be a novel non-invasive biomarker of antibody mediated rejection in renal transplantation.

  13. Comparison Of Blood Proteins And Some Hormonal Levels In Pregnant And Non-Pregnant Cows

    International Nuclear Information System (INIS)

    TEAMA, F.E.

    2010-01-01

    The aim of this study is to determine the changes in serum protein and its fractions by using electrophoresis in Holstein cows during different months of pregnancy in comparison with non-pregnant cows and to determine hormonal levels including T4, T3 and progesterone hormones. The samples were taken from 40 pregnant cows during deferent months and 10 non-pregnant cows. Significant decrease in the levels of total protein, albumin and globulin were observed in the third and late month of pregnancy than in mid pregnancy where the values were 6.5, 3.1 and 3.4 g/dl for early months and 6.5, 3.2 and 3.3 g/dl for late month as compared to the non-pregnant cows. Significant increase in α-1globulin was observed during months of pregnancy by about 33.3%. The decrease in the levels of α-2, β and γ-globulins were recorded by about 10%, 45.3% and 21.6%, respectively. A marked decrease in T4 hormone (5.0 μg/dl) was observed in pregnant cows than in non-pregnant ones (7.1 μg/dl). Also, a decreasing T3 level (169 ng/dl) was recorded as compared to non-pregnant cows (221 ng/dl). High significant increase in progesterone level was recorded in the mid pregnancy until reached the maximum value (49.94 ng/ml) at the 7 th month of pregnancy then declined (2.42 ng/ml) at the late month of pregnancy. In conclusion, during pregnancy of Holstein dairy cows, a decline in protein fractions and thyroid hormonal levels were recorded during different months as compared to non- pregnant cows. The opposite trend was observed in progesterone levels. The increasing progesterone level at the mid pregnancy indicated its importance in the continuation of pregnancy and maintenance of fetus against maternal rejection.

  14. Detection of interferon alpha protein reveals differential levels and cellular sources in disease.

    Science.gov (United States)

    Rodero, Mathieu P; Decalf, Jérémie; Bondet, Vincent; Hunt, David; Rice, Gillian I; Werneke, Scott; McGlasson, Sarah L; Alyanakian, Marie-Alexandra; Bader-Meunier, Brigitte; Barnerias, Christine; Bellon, Nathalia; Belot, Alexandre; Bodemer, Christine; Briggs, Tracy A; Desguerre, Isabelle; Frémond, Marie-Louise; Hully, Marie; van den Maagdenberg, Arn M J M; Melki, Isabelle; Meyts, Isabelle; Musset, Lucile; Pelzer, Nadine; Quartier, Pierre; Terwindt, Gisela M; Wardlaw, Joanna; Wiseman, Stewart; Rieux-Laucat, Frédéric; Rose, Yoann; Neven, Bénédicte; Hertel, Christina; Hayday, Adrian; Albert, Matthew L; Rozenberg, Flore; Crow, Yanick J; Duffy, Darragh

    2017-05-01

    Type I interferons (IFNs) are essential mediators of antiviral responses. These cytokines have been implicated in the pathogenesis of autoimmunity, most notably systemic lupus erythematosus (SLE), diabetes mellitus, and dermatomyositis, as well as monogenic type I interferonopathies. Despite a fundamental role in health and disease, the direct quantification of type I IFNs has been challenging. Using single-molecule array (Simoa) digital ELISA technology, we recorded attomolar concentrations of IFNα in healthy donors, viral infection, and complex and monogenic interferonopathies. IFNα protein correlated well with functional activity and IFN-stimulated gene expression. High circulating IFNα levels were associated with increased clinical severity in SLE patients, and a study of the cellular source of IFNα protein indicated disease-specific mechanisms. Measurement of IFNα attomolar concentrations by digital ELISA will enhance our understanding of IFN biology and potentially improve the diagnosis and stratification of pathologies associated with IFN dysregulation. © 2017 Rodero et al.

  15. Low level chemiluminescence measurement of the binding of 8-methoxypsoralen to proteins and lymphocytic surfaces

    International Nuclear Information System (INIS)

    Lange, B.

    1980-01-01

    Photochemotherapy with 8-methoxypsoralen (8-MOP) and longwave ultraviolet light is beneficial in such different disorders like psoriasis, lichen planus, and mykosis fungoides. In contrast to a widely accepted hypothesis 8-MOP does not solely bind to nucleic acid, but also to certain proteins. The mechanism of this binding as well as the precise binding area are unknown. Therefore the UV-provoked reactions of 8-MOP with a lipid mixture, a glucosaminoglycan solution, a protein solution, and lymphocyte suspensions, respectively were investigated using low level chemiluminescence (LLCL). It was found an 8-MOP concentration-dependent decrease of LLCL intensity in the lymphocyte suspensions (10 3 to 10 4 cells/μl). This effect is result of the diminution of the photoactive 8-MOP content of the solution. 8-MOP binds quickly and in the course of a free radical reaction to lymphocytic surfaces and coincidentally loses its potency to start LLCL-detectable free radical chain responses. (author)

  16. Inflammatory C-reactive protein and cytokine levels in asymptomatic people with chronic spinal cord injury.

    Science.gov (United States)

    Frost, Frederick; Roach, Mary Jo; Kushner, Irving; Schreiber, Peter

    2005-02-01

    To determine the relation between serologic markers of information and clinical characteristics of people with chronic spinal cord injury (SCI). Cross-sectional study. Academic medical center SCI outpatient clinic. Convenience sample of 37 men with chronic SCI and 10 healthy control subjects. Not applicable. Serum levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and C-reactive protein (CRP). The following results achieved statistical significance at P less than .05. Asymptomatic chronic SCI patients differed from referent controls with respect to serum CRP levels but not IL-6 or TNF-alpha. In SCI patients, higher levels of CRP correlated negatively with hemoglobin and albumin levels. A longer time since injury correlated with lower TNF-alpha values, whereas higher TNF-alpha levels correlated with higher serum albumin. Pressure ulcers and indwelling urinary catheters were associated with higher mean levels of CRP but not of the cytokines TNF-alpha and IL-6. Intermittent urinary catheterization was associated with lower levels of CRP when compared with other methods of bladder management. Asymptomatic people with long-term SCI, especially those with indwelling urinary catheters, showed serologic evidence of a systemic inflammatory state. There was no evidence of an elevation in proinflammatory cytokines. Detection of an ongoing systemic inflammatory response in apparently healthy people with indwelling urinary catheters and small skin ulcers further supports the aggressive pursuit of catheter-free voiding options and pressure ulcer healing.

  17. Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

    Science.gov (United States)

    Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

    2015-11-20

    The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

  18. Perinatal nicotine treatment induces transient increases in NACHO protein levels in the rat frontal cortex

    DEFF Research Database (Denmark)

    Wichern, Franziska; Jensen, Majbrit M; Christensen, Ditte Z

    2017-01-01

    The nicotinic acetylcholine receptor (nAChR) regulator chaperone (NACHO) was recently identified as an important regulator of nAChR maturation and surface expression. Here we show that NACHO levels decrease during early postnatal development in rats. This decrease occurs earlier and to a greater...... degree in the frontal cortex (FC) compared with the hippocampus (HIP). We further show that rats exposed to nicotine during pre- and postnatal development exhibit significantly higher NACHO levels in the FC at postnatal day (PND) 21, but not at PND60. Repeated exposure to nicotine selectively during...... a single exposure to a combination of nicotine and the type II α7 nAChR positive allosteric modulator (PAM) PNU-120596, but not the type I PAM AVL-3288. These findings suggest that exposure to nAChR agonism affects NACHO protein levels, and that this effect is more pronounced during pre- or early postnatal...

  19. Carvacrol attenuates serum levels of total protein, phospholipase A2 and histamine in asthmatic guinea pig

    Directory of Open Access Journals (Sweden)

    Mohammad Hossein Boskabady

    2016-11-01

    Full Text Available Objective: Pharmacological effects of carvacrol such as its anti-inflammatory activities have been shows. In this study the effects of carvacrol on serum levels of total protein (TP, phospholipase A2 (PLA2 and histamine in sensitized guinea pigs was evaluated. Materials and Methods: Sensitized guinea pigs were given drinking water alone (group S, drinking water containing three concentrations of carvacrol (40, 80 and 160 µg/ml or dexamethasone. Serum levels of TP, PLA2 and histamine were examined I all sensitized groups as well as a non-sensitized control group (n=6 for each group. Results: In sensitized animals, serum levels of TP, PLA2 and histamine were significantly increased compared to control animals (p

  20. High order quaternary arrangement confers increased structural stability to Brucella Spp. lumazine synthase

    Energy Technology Data Exchange (ETDEWEB)

    Zylberman, V.; Craig, P.O.; Klinke, S.; Cauerhff, A.; Goldbaum, F.A. [Instituto Leloir, Buenos Aires (Argentina); Braden, B.C. [Bowie State Univ., Maryland (United States)

    2004-07-01

    The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18 kDa monomers, each extensively contacting neighboring monomers. The icosahedral structure consists of 60 LS monomers arranged as twelve pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella spp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedral enzymes. This unusual divergence prompted to further investigate on its quaternary arrangement. In the present work, we demonstrate by means of solution Light Scattering and X-ray structural analyses that BLS assembles as a very stable dimer of pentamers representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein, and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 deg C compared to pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. (author)

  1. High order quaternary arrangement confers increased structural stability to Brucella Spp. lumazine synthase

    International Nuclear Information System (INIS)

    Zylberman, V.; Craig, P.O.; Klinke, S.; Cauerhff, A.; Goldbaum, F.A.; Braden, B.C.

    2004-01-01

    The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18 kDa monomers, each extensively contacting neighboring monomers. The icosahedral structure consists of 60 LS monomers arranged as twelve pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella spp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedral enzymes. This unusual divergence prompted to further investigate on its quaternary arrangement. In the present work, we demonstrate by means of solution Light Scattering and X-ray structural analyses that BLS assembles as a very stable dimer of pentamers representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein, and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 deg C compared to pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. (author)

  2. Influence of different levels of concentrate and ruminally undegraded protein on digestive variables in beef heifers.

    Science.gov (United States)

    Pina, D S; Valadares Filho, S C; Tedeschi, L O; Barbosa, A M; Valadares, R F D

    2009-03-01

    This experiment evaluated the effect of 2 levels of diet concentrate (20 and 40% of DM) and 2 levels of ruminally undegraded protein (RUP: 25 and 40% of CP) on nutrient intake, total and partial apparent nutrient digestibility, microbial protein synthesis, and ruminal and physiological variables. Eight Nellore heifers (233 +/- 14 kg of BW) fitted with ruminal, abomasal, and ileal cannulas were used. The animals were held in individual sheltered pens of approximately 15 m(2) and fed twice daily at 0800 and 1600 h for ad libitum intake. Heifers were allocated in two 4 x 4 Latin square designs, containing 8 heifers, 4 experimental periods, and 4 treatments in a 2 x 2 factorial arrangement. All statistical analyses were performed using PROC MIXED of SAS. Titanium dioxide (TiO(2)) and chromic oxide (Cr(2)O(3)) were used to estimate digesta fluxes and fecal excretion. Purine derivative (PD) excretion and abomasal purine bases were used to estimate the microbial N (MN) synthesis. No significant interaction (P > 0.10) between dietary levels of RUP and concentrate was observed. There was no effect of treatment (P = 0.24) on DMI. Both markers led to the same estimates of fecal, abomasal, and ileal DM fluxes, and digestibilities of DM and individual nutrients. Ruminal pH was affected by sampling time (P RUP, whereas a quadratic effect (P RUP. The higher level of dietary concentrate led to greater MN yield regardless of the level of RUP. The MN yield and the efficiency of microbial yield estimated from urinary PD excretion produced greater (P RUP and concentrate were observed for ruminal and digestive parameters. Neither RUP nor concentrate level affected DMI. Titanium dioxide showed to be similar to Cr(2)O(3) as an external marker to measure digestibility and nutrient fluxes in cattle.

  3. Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer.

    LENUS (Irish Health Repository)

    Cathcart, Mary-Clare

    2012-02-01

    BACKGROUND: Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. METHODS: PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. RESULTS: PGIS expression was reduced\\/absent in human NSCLC protein samples (P < .0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P = .004) and in male patients (P < .05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P < .001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. CONCLUSIONS: PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC.

  4. The crystal structure of human GDP-L-fucose synthase.

    Science.gov (United States)

    Zhou, Huan; Sun, Lihua; Li, Jian; Xu, Chunyan; Yu, Feng; Liu, Yahui; Ji, Chaoneng; He, Jianhua

    2013-09-01

    Human GDP-l-fucose synthase, also known as FX protein, synthesizes GDP-l-fucose from its substrate GDP-4-keto-6-deoxy-d-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. In this paper, the first crystal structure of human FX protein was determined at 2.37 Å resolution. The asymmetric unit of the crystal structure contains four molecules which form two homodimers. Each molecule consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain. Compared with the Escherichia coli GDP-l-fucose synthase, the overall structures of these two enzymes have four major differences. There are four loops in the structure of human FX protein corresponding to two α-helices and two β-sheets in that of the E. coli enzyme. Besides, there are seven different amino acid residues binding with NAPDH comparing human FX protein with that from E. coli. The structure of human FX reveals the key catalytic residues and could be useful for the design of drugs for the treatment of inflammation, auto-immune diseases, and possibly certain types of cancer.

  5. Serum Levels of Surfactant Proteins in Patients with Combined Pulmonary Fibrosis and Emphysema (CPFE.

    Directory of Open Access Journals (Sweden)

    Andriana I Papaioannou

    Full Text Available Emphysema and idiopathic pulmonary fibrosis (IPF present either per se or coexist in combined pulmonary fibrosis and emphysema (CPFE. Serum surfactant proteins (SPs A, B, C and D levels may reflect lung damage. We evaluated serum SP levels in healthy controls, emphysema, IPF, and CPFE patients and their associations to disease severity and survival.122 consecutive patients (31 emphysema, 62 IPF, and 29 CPFE and 25 healthy controls underwent PFTs, ABG-measurements, 6MWT and chest HRCT. Serum levels of SPs were measured. Patients were followed-up for 1-year.SP-A and SP-D levels differed between groups (p = 0.006 and p<0.001 respectively. In post-hoc analysis, SP-A levels differed only between controls and CPFE (p<0.05 and CPFE and emphysema (p<0.05. SP-D differed between controls and IPF or CPFE (p<0.001 for both comparisons. In IPF SP-B correlated to pulmonary function while SP-A, correlated to the Composite Physiological Index (CPI. Controls current smokers had higher SP-A and SP-D levels compared to non-smokers (p = 0.026 and p = 0.023 respectively. SP-D levels were higher in CPFE patients with extended emphysema (p = 0.042. In patients with IPF, SP-B levels at the upper quartile of its range (≥26 ng/mL presented a weak association with reduced survival (p = 0.05.In conclusion, serum SP-A and SP-D levels were higher where fibrosis exists or coexists and related to disease severity, suggesting that serum SPs relate to alveolar damage in fibrotic lungs and may reflect either local overproduction or overleakage. The weak association between high levels of SP-B and survival needs further validation in clinical trials.

  6. Intrahepatic cholangiocarcinoma prognostic determination using pre-operative serum C-reactive protein levels

    International Nuclear Information System (INIS)

    Lin, Zi-Ying; Liang, Zhen-Xing; Zhuang, Pei-Lin; Chen, Jie-Wei; Cao, Yun; Yan, Li-Xu; Yun, Jing-Ping; Xie, Dan; Cai, Mu-Yan

    2016-01-01

    Serum C-reactive protein (CRP), an acute inflammatory response biomarker, has been recognized as an indicator of malignant disease progression. However, the prognostic significance of CRP levels collected before tumor removal in intrahepatic cholangiocarcinoma requires further investigation. We sampled the CRP levels in 140 patients with intrahepatic cholangiocarcinoma who underwent hepatectomies with regional lymphadenectomies between 2006 and 2013. A retrospective analysis of the clinicopathological data was performed. We focused on the impact of serum CRP on the patients’ cancer-specific survival and recurrence-free survival rates. High levels of preoperative serum CRP were significantly associated with well-established clinicopathologic features, including gender, advanced tumor stage, and elevated carcinoembryonic antigen and carbohydrate antigen 19-9 levels (P < 0.05). Univariate analysis demonstrated a significant association between high levels of serum CRP and adverse cancer-specific survival (P = 0.001) and recurrence-free survival (P < 0.001). In patients with stage I/II intrahepatic cholangiocarcinoma, the serum CRP level was a prognostic indicator for cancer-specific survival. In patients with stage I/II or stage III/IV, the serum CRP level was a prognostic indicator for recurrence-free survival (P < 0.05). Additionally, multivariate analysis identified serum CRP level in intrahepatic cholangiocarcinoma as an independent prognostic factor (P < 0.05). We confirmed a significant association of elevated pre-operative CRP levels with poor clinical outcomes for the tested patients with intrahepatic cholangiocarcinoma. Our results indicate that the serum CRP level may represent a useful factor for patient stratification in intrahepatic cholangiocarcinoma management

  7. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

    2011-12-31

    'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

  8. Effect of Hypergravity on the Level of Heat Shock Proteins 70 and 90 in Pea Seedlings

    Science.gov (United States)

    Kozeko, Liudmyla; Kordyum, Elizabeth

    2009-01-01

    Exposure to hypergravity induces significant changes in gene expression of plants which are indicative of stress conditions. A substantial part of the general stress response is up-regulation of heat shock proteins (Hsp) which function as molecular chaperones. The objective of this research was to test the possible changes in the Hsp70 and Hsp90 level in response to short-term hypergravity exposure. In this study 5-day-old etiolated pea seedlings were exposed to centrifuge-induced hypergravity (3-14 g) for 15 min and 1 h and a part of the seedlings was sampled at 1.5 and 24 h after the exposures. Western blot analysis showed time-dependent changes in Hsp70 and Hsp90 levels: an increase under hypergravity and a tendency towards recovery of the normal content during re-adaptation. The quantity and time of their expression was correlated with the g-force level. These data suggest that short-term hypergravity acts as a stress which could increase the risk of protein denaturation and aggregation. Molecular chaperons induced during the stress may have an essential role in counteracting this risk.

  9. Increasing levels of crude protein in multiple supplements for grazing beef heifers in rainy season

    Directory of Open Access Journals (Sweden)

    Lívia Vieira de Barros

    2015-06-01

    Full Text Available The objective was to evaluate the effect of multiple supplements with differents levels of crude protein (CP or mineral supplements on the nutritional parameters and performance of beef heifers grazing Uruchloa decumbens in the rainy season. A complete random design was employed. The treatments were made up of increasing levels of CP in the multiple supplements and a control treatment (MM in which animals were offered only mineral mixture. Multiple supplements contained 17; 30; 43 and 56% of CP, for treatments CP17; CP30; CP43 and CP56, respectively. Average daily gain (ADG (g was 447.7; 554.6; 638.4; 587.9; 590.4, for treatments MM, CP17; CP30; CP43 and CP56, respectively. A quadratic effect of the levels of crude protein was found (p< 0.10 on ADG. A greater intake of dry matter (DM, organic matter (OM, CP, ether extract (EE, non-fibrous carbohydrates (NFC, total digestible nutrients (TDN, and digested dry matter (p< 0.10 was found in animals supplemented with multiple supplements. Multiple supplements increased the apparent digestibility coefficient of DM, CP, EE and NFC. Supply of multiple multiple supplements for heifers grazing in medium to high quality pastures in the rainy season improves the performance of the animals.

  10. Plasma levels of selenium-containing proteins in Inuit adults from Nunavik.

    Science.gov (United States)

    Achouba, Adel; Dumas, Pierre; Ouellet, Nathalie; Lemire, Mélanie; Ayotte, Pierre

    2016-11-01

    Selenium (Se) is highly abundant in marine foods traditionally consumed by Inuit of Nunavik (Northern Quebec, Canada) and accordingly, their Se intake is among the highest in the world. However, little is known regarding the biological implications of this high Se status in this Arctic indigenous population. We used a method combining affinity chromatography and inductively coupled plasma-mass spectrometry with quantification by post-column isotope dilution to determine total Se levels and concentrations of Se-containing proteins in archived plasma samples of Inuit adults who participated to the 2004 Nunavik Inuit Health Survey (N = 852). Amounts of mercury (Hg) associated with Se-containing proteins were also quantified. Results show that glutathione peroxidase 3 (GPx3), selenoprotein P (SelP) and selenoalbumin (SeAlb) represented respectively 25%, 52% and 23% of total plasma Se concentrations. In addition, small amounts of Hg co-eluted with each Se-containing protein and up to 50% of plasma Hg was associated to SelP. Total plasma Se concentrations (median = 139 μg L− 1; interquartile range (IQR) = 22.7 μg L− 1) were markedly lower and less variable than whole blood Se concentration (median = 261 μg L− 1, IQR = 166 μg L− 1). A non linear relation was observed between whole blood Se and plasma Se levels, with plasma Se concentrations leveling off at approximately 200 μg L− 1, whereas 16% and 3% of individuals exhibited whole blood concentrations higher than 500 μg L− 1 and 1000 μg L− 1, respectively. In contrast, a linear relationship was previously reported in communities consuming Brazil nuts which are rich Se, mainly present as selenomethionine. This suggests that a different selenocompound, possibly selenoneine, is present in the Arctic marine food chain and accumulates in the blood cellular fraction of Inuit.

  11. Effects of secretagogues on ATP levels and protein carboxyl methylation in rat brain synaptosomes

    International Nuclear Information System (INIS)

    Bjorndahl, J.M.; Rutledge, C.O.

    1986-01-01

    The influence of various substances which are known to alter free intracellular calcium concentrations on protein carboxyl methyltransferase (PCM) activity was investigated in rat brain synaptosomes. The synaptosomes were labeled with L-[ 3 H]methionine and the 3 H-methyl esters of proteins were formed from the methyl donor S-[ 3 H]adenosyl-L-methionine ([ 3 H]AdoMet). The calcium ionophore A23187 and ouabain decreased PCM activity and the decrease produced by A23187 was antagonized by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and MnCl 2 . On the other hand, ruthenium red, an inhibitor of calcium uptake, stimulated PCM activity. These data suggest that PCM activity is inversely related to the free cytoplasmic calcium concentration. Veratridine, A23187 and elevated potassium ions decreased the levels of ATP and [ 3 H]AdoMet. The A23187-mediated decrease in ATP levels and the reduced [ 3 H]AdoMet formation was antagonized by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and MnCl 2 . Inhibition of metabolic activity of the synaptosomes by NaCN led to: decreased ATP levels; inhibition of [3H]AdoMet formation; and inhibition of PCM activity. These data suggest that the decrease in protein methylation produced by secretagogues is associated with an increase in the concentration of free intracellular calcium which results in a decrease in the metabolically active pool of ATP. This leads to a decreased rate of AdoMet formation, a cosubstrate for PCM and a resultant decrease in PCM activity

  12. Macrocyclic peptides decrease c-Myc protein levels and reduce prostate cancer cell growth.

    Science.gov (United States)

    Mukhopadhyay, Archana; Hanold, Laura E; Thayele Purayil, Hamsa; Gisemba, Solomon A; Senadheera, Sanjeewa N; Aldrich, Jane V

    2017-08-03

    The oncoprotein c-Myc is often overexpressed in cancer cells, and the stability of this protein has major significance in deciding the fate of a cell. Thus, targeting c-Myc levels is an attractive approach for developing therapeutic agents for cancer treatment. In this study, we report the anti-cancer activity of the macrocyclic peptides [D-Trp]CJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp]) and the natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]). [D-Trp]CJ-15,208 reduced c-Myc protein levels in prostate cancer cells and decreased cell proliferation with IC 50 values ranging from 2.0 to 16 µM in multiple PC cell lines. [D-Trp]CJ-15,208 induced early and late apoptosis in PC-3 cells following 48 hours treatment, and growth arrest in the G2 cell cycle phase following both 24 and 48 hours treatment. Down regulation of c-Myc in PC-3 cells resulted in loss of sensitivity to [D-Trp]CJ-15,208 treatment, while overexpression of c-Myc in HEK-293 cells imparted sensitivity of these cells to [D-Trp]CJ-15,208 treatment. This macrocyclic tetrapeptide also regulated PP2A by reducing the levels of its phosphorylated form which regulates the stability of cellular c-Myc protein. Thus [D-Trp]CJ-15,208 represents a new lead compound for the potential development of an effective treatment of prostate cancer.

  13. Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level, Genotype and Developmental Stages.

    Science.gov (United States)

    Liu, Yingying; Li, Fengna; Kong, Xiangfeng; Tan, Bie; Li, Yinghui; Duan, Yehui; Blachier, François; Hu, Chien-An A; Yin, Yulong

    2015-01-01

    Muscle growth is regulated by the homeostatic balance of the biosynthesis and degradation of muscle proteins. To elucidate the molecular interactions among diet, pig genotype, and physiological stage, we examined the effect of dietary protein concentration, pig genotype, and physiological stages on amino acid (AA) pools, protein deposition, and related signaling pathways in different types of skeletal muscles. The study used 48 Landrace pigs and 48 pure-bred Bama mini-pigs assigned to each of 2 dietary treatments: lower/GB (Chinese conventional diet)- or higher/NRC (National Research Council)-protein diet. Diets were fed from 5 weeks of age to respective market weights of each genotype. Samples of biceps femoris muscle (BFM, type I) and longissimus dorsi muscle (LDM, type II) were collected at nursery, growing, and finishing phases according to the physiological stage of each genotype, to determine the AA concentrations, mRNA levels for growth-related genes in muscles, and protein abundances of mechanistic target of rapamycin (mTOR) signaling pathway. Our data showed that the concentrations of most AAs in LDM and BFM of pigs increased (Prelated AA, including Met, Phe, Tyr, Pro, and Ser, compared with Landrace pigs. The mRNA levels for myogenic determining factor, myogenin, myocyte-specific enhancer binding factor 2 A, and myostatin of Bama mini-pigs were higher (P<0.05) than those of Landrace pigs, while total and phosphorylated protein levels for protein kinase B, mTOR, and p70 ribosomal protein S6 kinases (p70S6K), and ratios of p-mTOR/mTOR, p-AKT/AKT, and p-p70S6K/p70S6K were lower (P<0.05). There was a significant pig genotype-dependent effect of dietary protein on the levels for mTOR and p70S6K. When compared with the higher protein-NRC diet, the lower protein-GB diet increased (P<0.05) the levels for mTOR and p70S6K in Bama mini-pigs, but repressed (P<0.05) the level for p70S6K in Landrace pigs. The higher protein-NRC diet increased ratio of p-mTOR/mTOR in

  14. Pregnancy-associated plasma protein-a levels in individuals with and without coronary artery disease

    International Nuclear Information System (INIS)

    Khan, N.U.; Khan, F.A.; Khan, D.A.; Asim, N.

    2011-01-01

    Objective: To compare pregnancy-associated plasma protein-A (PAPP-A) levels in individuals with and without coronary artery disease (CAD). Study Design: Cross-sectional comparative study. Place and Duration of Study: Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, in collaboration with Armed Forces Institute of Cardiology (AFIC), from September 2008 to March 2010. Methodology: One hundred and twenty five (125) individuals both male and female were included in the study. Blood for PAPP-A and lipid profile was collected, just before angiography. On the basis of angiography, the individuals were divided into those with and without CAD. PAPP-A was analyzed by using Diagnostic System Laboratories (DSL) Enzyme Linked Immunosorbent Assay (ELISA) kit and reading was taken by ELISA reader. Lipid profile was determined on automated analyzers Selectra-2 and Vitros 5.1. Results: Amongst the 125 individuals, 41 individuals were without CAD whereas 84 individuals were having CAD. Mean PAPP-A levels were 0.74 +- 0.35 mIU/L in those without CAD whereas mean PAPP-A levels in those with CAD were 1.35 +- 0.57 mIU/L. The difference between the two groups was statistically significant (p < 0.001). A PAPP-A cut off level of 0.85 mIU/L had a sensitivity and specificity of 78% and 70% respectively for diagnosing atherosclerotic CAD. Conclusion: PAPP-A is a potentially relevant marker of the presence and extent of coronary atherosclerosis as its levels are elevated in CAD as compared to individuals without CAD. Pregnancy-associated plasma protein-A. (author)

  15. Geranylgeranyl diphosphate synthase from Scoparia dulcis and Croton sublyratus. Plastid localization and conversion to a farnesyl diphosphate synthase by mutagenesis.

    Science.gov (United States)

    Sitthithaworn, W; Kojima, N; Viroonchatapan, E; Suh, D Y; Iwanami, N; Hayashi, T; Noji, M; Saito, K; Niwa, Y; Sankawa, U

    2001-02-01

    cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene-producing plants, Scoparia dulcis and Croton sublyratus, have been isolated using the homology-based polymerase chain reaction (PCR) method. Both clones contained highly conserved aspartate-rich motifs (DDXX(XX)D) and their N-terminal residues exhibited the characteristics of chloroplast targeting sequence. When expressed in Escherichia coli, both the full-length and truncated proteins in which the putative targeting sequence was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to produce geranylgeranyl diphosphate (GGPP). The structural factors determining the product length in plant GGPPSs were investigated by constructing S. dulcis GGPPS mutants on the basis of sequence comparison with the first aspartate-rich motif (FARM) of plant farnesyl diphosphate synthase. The result indicated that in plant GGPPSs small amino acids, Met and Ser, at the fourth and fifth positions before FARM and Pro and Cys insertion in FARM play essential roles in determination of product length. Further, when a chimeric gene comprised of the putative transit peptide of the S. dulcis GGPPS gene and a green fluorescent protein was introduced into Arabidopsis leaves by particle gun bombardment, the chimeric protein was localized in chloroplasts, indicating that the cloned S. dulcis GGPPS is a chloroplast protein.

  16. Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum).

    Science.gov (United States)

    Yang, Chang-Qing; Wu, Xiu-Ming; Ruan, Ju-Xin; Hu, Wen-Li; Mao, Yin-Bo; Chen, Xiao-Ya; Wang, Ling-Jian

    2013-12-01

    Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Growth Response of White Shrimp (Litopenaeus Vannamei) Reared in Low Salinity Medium, Fed Different Protein and Calcium Levels

    OpenAIRE

    Kaligis, Erly

    2015-01-01

    The white shrimp (Litopenaeus vannamei) has been an important commercial shrimp species in Indonesia. This species is tolerance to low salinity therefore, it is important to develop its aquaculture. The purpose of this study was to study the effect of protein and calcium levels in diet on growth performance of the white shrimp post larvae. A factorial experiment at three levels of dietary protein (25, 35, 45%) and three levels of calcium (0, 2, 4%) with three replicates were used in this expe...

  18. Homocystinuria due to cystathionine beta synthase deficiency

    Directory of Open Access Journals (Sweden)

    Rao T

    2008-01-01

    Full Text Available A two year-old male child presented with cutis marmorata congenita universalis, brittle hair, mild mental retardation, and finger spasms. Biochemical findings include increased levels of homocysteine in the blood-106.62 µmol/L (normal levels: 5.90-16µmol/L. Biochemical tests such as the silver nitroprusside and nitroprusside tests were positive suggesting homocystinuria. The patient was treated with oral pyridoxine therapy for three months. The child responded well to this therapy and the muscle spasms as well as skin manifestations such as cutis marmorata subsided. The treatment is being continued; the case is reported here because of its rarity. Homocysteinuria arising due to cystathionine beta-synthase (CBS deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine It manifests itself in vascular, central nervous system, cutaneous, and connective tissue disturbances and phenotypically resembles Marfan′s syndrome. Skin manifestations include malar flush, thin hair, and cutis reticulata / marmorata.

  19. Investigation of New Morpholino Oligomers to Increase Survival Motor Neuron Protein Levels in Spinal Muscular Atrophy.

    Science.gov (United States)

    Ramirez, Agnese; Crisafulli, Sebastiano G; Rizzuti, Mafalda; Bresolin, Nereo; Comi, Giacomo P; Corti, Stefania; Nizzardo, Monica

    2018-01-06

    Spinal muscular atrophy (SMA) is an autosomal-recessive childhood motor neuron disease and the main genetic cause of infant mortality. SMA is caused by deletions or mutations in the survival motor neuron 1 ( SMN1 ) gene, which results in SMN protein deficiency. Only one approved drug has recently become available and allows for the correction of aberrant splicing of the paralogous SMN2 gene by antisense oligonucleotides (ASOs), leading to production of full-length SMN protein. We have already demonstrated that a sequence of an ASO variant, Morpholino (MO), is particularly suitable because of its safety and efficacy profile and is both able to increase SMN levels and rescue the murine SMA phenotype. Here, we optimized this strategy by testing the efficacy of four new MO sequences targeting SMN2 . Two out of the four new MO sequences showed better efficacy in terms of SMN protein production both in SMA induced pluripotent stem cells (iPSCs) and SMAΔ7 mice. Further, the effect was enhanced when different MO sequences were administered in combination. Our data provide an important insight for MO-based treatment for SMA. Optimization of the target sequence and validation of a treatment based on a combination of different MO sequences could support further pre-clinical studies and the progression toward future clinical trials.

  20. Dependence of intestinal amino acid uptake on dietary protein or amino acid levels

    International Nuclear Information System (INIS)

    Karasov, W.H.; Solberg, D.H.; Diamond, J.M.

    1987-01-01

    To understand how intestinal amino acid (AA) transport is regulated by dietary substrate levels, the authors measured uptake of seven radioactively-labelled AAs and glucose across the jejunal brush-border membrane of mice kept on one of three isocaloric rations differing in nitrogen content. In the high-protein ration, uptake increased by 77-81% for the nonessential, less toxic AAs, proline, and aspartate but only by 32-61% for the more toxic essential AAs tested. In the nitrogen-deficient ration, uptake decreased for the nonessential aspartate and proline but stayed constant or increased for essential AAs and for the nonessential alanine. These patterns imply independent regulation of the intestine's various AA transporters. With decreasing dietary AA (or protein), the imino acid and acidic AA private transporters are repressed, while activities of the basic AA transporter and the neutral AA public transporter decrease to an asymptote or else go through a minimum. These regulatory patterns can be understood as a compromise among conflicting constraints imposed by protein's multiple roles as a source of calories, nitrogen, and essential AAs and by the toxicity of essential AAs at high concentrations

  1. Evaluation of behaviour in stabled draught horse foals fed diets with two protein levels.

    Science.gov (United States)

    Sartori, C; Guzzo, N; Normando, S; Bailoni, L; Mantovani, R

    2017-01-01

    The present work is aimed at evaluating the behaviour of Italian Heavy Draught Horse (IHDH) foals reared in semi-covered stables and fed two isoenergetic total mixed rations with different dietary protein levels (13.2% and 10.6% of CP on dry matter). The study was prompted by the restrictions for nitrate emissions in farms of the European Nitrate Directive. One suggested solution is to reduce dietary protein while maintaining normal performance and welfare, but there is a lack of literature in studies of horses. The behaviours of 20 foals of 437±60 kg of BW, aged 379±37 days and stabled in four pens by sex (S) and diet (D) were video recorded and analysed to build a suitable ethogram including 18 behaviours in six categories: ingestion, resting, maintenance, movement, social activities, other. The percentage of the daily time spent in each behavioural category and single behaviours was analysed via a single traits GLM including S, D and their interaction. Daily activity was consistent with existing literature: foals spent about 33% of the day in ingestion activities and 41% in resting, whereas social interactions constituted 8% of the time and individual maintenance draught breeds for foals in both dietary groups, a result that suggests the maintenance of well-being after dietary protein reduction. This result, together with the findings of a companion study showing no changes in growth performances of foals, showed that a reduction of CP in foal diet is reconcilable with the maintenance of performance and welfare.

  2. Effect of dietary protein quality and feeding level on milk secretion and mammary protein synthesis in the rat

    International Nuclear Information System (INIS)

    Sampson, D.A.; Jansen, G.R.

    1985-01-01

    Protein synthesis was studied in mammary tissue of rats fed diets deficient in protein quality and/or restricted in food intake throughout gestation and lactation. Diets containing 25% wheat gluten (WG), wheat gluten plus lysine and threonine (WGLT), or casein (C) were pair-fed from conception until day 15 of lactation at 100% or 85% of WG ad libitum consumption (PF100 and PF85, respectively). A seventh group was fed C ad libitum. Rates of protein synthesis were measured in vivo at day 15 of lactation from incorporation of [3- 3 H]phenylalanine. At both PF100 and PF85, fractional and absolute rates of mammary gland protein synthesis were two- to three-fold higher in rats fed C than in those fed WG. Pup weights showed similar treatment effects. Both mammary protein synthesis rates and pup weights were significantly higher in rats fed C at PF85 than rats fed WG ad libitum. Food restriction from PF100 to PF85 depressed pup weights and mammary protein synthesis rates in rats fed WGLT, but had no effect in rats fed WG. These results demonstrate that when food intake is restricted, improvement of protein quality of the maternal diet increases milk output in the rat in association with increased rates of mammary protein synthesis

  3. Present perspectives on the automated classification of the G-protein coupled receptors (GPCRs) at the protein sequence level

    DEFF Research Database (Denmark)

    Davies, Matthew N; Gloriam, David E; Secker, Andrew

    2011-01-01

    The G-protein coupled receptors--or GPCRs--comprise simultaneously one of the largest and one of the most multi-functional protein families known to modern-day molecular bioscience. From a drug discovery and pharmaceutical industry perspective, the GPCRs constitute one of the most commercially an...

  4. [High-level expression of heterologous protein based on increased copy number in Saccharomyces cerevisiae].

    Science.gov (United States)

    Zhang, Xinjie; He, Peng; Tao, Yong; Yang, Yi

    2013-11-04

    High-level expression system of heterologous protein mediated by internal ribosome entry site (IRES) in Saccharomyces cerevisiae was constructed, which could be used for other applications of S. cerevisiae in metabolic engineering. We constructed co-expression cassette (promoter-mCherry-TIF4631 IRES-URA3) containing promoters Pilv5, Padh2 and Ptdh3 and recombined the co-expression cassette into the genome of W303-1B-A. The URA3+ transformants were selected. By comparing the difference in the mean florescence value of mCherry in transformants, the effect of three promoters was detected in the co-expression cassette. The copy numbers of the interested genes in the genome were determined by Real-Time PCR. We analyzed genetic stability by continuous subculturing transformants in the absence of selection pressure. To verify the application of co-expression cassette, the ORF of mCherry was replaced by beta-galactosidase (LACZ) and xylose reductase (XYL1). The enzyme activities and production of beta-galactosidase and xylose reductase were detected. mCherry has been expressed in the highest-level in transformants with co-expression cassette containing Pilv5 promoter. The highest copy number of DNA fragment integrating in the genome was 47 in transformants containing Pilv5. The engineering strains showed good genetic stability. Xylose reductase was successfully expressed in the co-expression cassette containing Pilv5 promoter and TIF4631 IRES. The highest enzyme activity was 0. 209 U/mg crude protein in the transformants WIX-10. Beta-galactosidase was also expressed successfully. The transformants that had the highest enzyme activity was WIL-1 and the enzyme activity was 12.58 U/mg crude protein. The system mediated by Pilv5 promoter and TIF4631 IRES could express heterologous protein efficiently in S. cerevisiae. This study offered a new strategy for expression of heterologous protein in S. cerevisiae and provided sufficient experimental evidence for metabolic engineering

  5. Expression, purification and preliminary crystallographic analysis of sucrose phosphate synthase (SPS) from Halothermothrix orenii

    International Nuclear Information System (INIS)

    Huynh, Frederick; Tan, Tien-Chye; Swaminathan, Kunchithapadam; Patel, Bharat K. C.

    2004-01-01

    The first crystallographic study of a sucrose phosphate synthase from H. orenii, an organism that is both thermophilic and halophilic, is reported. The protein crystal diffracts X-rays to 3.01 Å. This is the first report of the crystallization of a sucrose phosphate synthase (SPS; EC 2.4.1.14). It also constitutes the first study of a sucrose phosphate synthase from a non-photosynthetic thermohalophilic anaerobic bacterium, Halothermothrix orenii. The purified recombinant spsA protein has been crystallized in the monoclinic space group C2, with unit-cell parameters a = 154.2, b = 47.9, c = 72.3 Å, β = 103.16°, using the hanging-drop vapour-diffusion method. The crystal diffracts X-rays to a resolution limit of 3.01 Å. Heavy-metal and halide-soaking trials are currently in progress to solve the structure

  6. The effect of protein-energy levels dietary on Kacang goats performances

    Directory of Open Access Journals (Sweden)

    MuchJi Martawidjaja

    1999-10-01

    Full Text Available An experiment was done to evaluate the protein-energy requirement for growing Kacang goats. Twelve males and 18 female goats, seven to eight months old were used in this study and randomized into three treatment groups, with four and six animals each, and were kept in individual pens. The treatments used were: R1= Elephant grass (E.G. + concentrate C1 (21% CP; 3.9 Mcal GE/kg, R2 = E.G. + concentrate C2 (17% CP; 3.7 Mcal GE/kg, and R3 = E.G. + concentrate C3 (12% CP; 3.5 Mcal GE/kg, respectively. Fresh Elephant grass was offered in restricted, and concentrate was offered at 3% of body weight. The experiment was carried out for 12 weeks. Data were analysed by using factorial completely randomized design 2x3 (3 rations and 2 sexes. Parameters measured were: feed intake; average daily gain and feed conversion. The results indicated that among treatments there was no significant difference on dry matter (DM and gross energy (GE intake (P>0.05, but crude protein (CP intake of R1 was 23,6% higher than treatment R2; treatment R2 was 38.1% higher than R3 (P0.05, but treatment R1 was 36.9% and significantly higher than R3 (P0.05, but ration R1 was more efficient than R3 (P0.05. It was concluded that protein intake and average daily gain were increased, and feed conversion was more efficient as the crude protein-energy levels increased in the ration. Feed intake and average daily gain of male goats were higher and feed conversion was more efficient than the female goats.

  7. The Prognostic Value of C-Reactive Protein Serum Levels in Patients with Uterine Leiomyosarcoma.

    Directory of Open Access Journals (Sweden)

    Richard Schwameis

    Full Text Available C-reactive protein (CRP has previously been shown to serve as a prognostic parameter in women with gynecologic malignancies. Due to the lack of valid prognostic markers for uterine leiomyosarcoma (ULMS this study set out to investigate the value of pre-treatment CRP serum levels as prognostic parameter.Data of women with ULMS were extracted from databases of three Austrian centres for gynaecologic oncology. Pre-treatment CRP serum levels were measured and correlated with clinico-pathological parameters. Univariate and multivariable survival analyses were performed.In total, 53 patients with ULMS were included into the analysis. Mean (SD CRP serum level was 3.46 mg/dL (3.96. Solely, an association between pre-treatment CRP serum levels and tumor size (p = 0.04 but no other clinic-pathologic parameter such as tumor stage (p = 0.16, or histological grade (p = 0.07, was observed. Univariate and multivariable survival analyses revealed that CRP serum levels (HR 2.7 [1.1-7.2], p = 0.037 and tumor stage (HR 6.1 [1.9-19.5], p = 0.002 were the only independent prognostic factors for overall survival (OS in patients with ULMS. Patients with high pre-treatment CRP serum levels showed impaired OS compared to women with low levels (5-year-OS rates: 22.6% and 52.3%, p = 0.007.High pre-treatment CRP serum levels were independently associated with impaired prognosis in women with ULMS and might serve as a prognostic parameter in these patients.

  8. Elevated urine levels of heparin-binding protein in children with urinary tract infection.

    Science.gov (United States)

    Kjölvmark, Charlott; Akesson, Per; Linder, Adam

    2012-08-01

    Urinary tract infection (UTI) is a common infection diagnosis in children, and efficient diagnosis and treatment are important to avoid serious complications. In this study we investigated whether urinary levels of neutrophil-derived heparin-binding protein (HBP) can be used as a marker of UTI in children. These results were compared to those of dipstick analysis, interleukin-6 (IL-6) analysis in urine, and bacterial culturing. Seventy-eight children aged 0-18 years with fever and/or symptoms indicating UTI were enrolled in a prospective consecutive study. Urine samples were cultured and analyzed with dipstick, and concentrations of HBP and IL-6 were measured. Fifteen patients were classified as having UTI, 30 patients had fever but were diagnosed with a non-urinary tract infection, and 33 patients had neither UTI nor fever. Using a urine HBP (U-HBP) cut-off level of 32 ng/mL, the sensitivity and specificity for detecting UTI were 93.3 and 90.3 %, respectively. Receiver operating characteristic curves demonstrated that U-HBP levels were a higher specificity indicator of UTI than urine white blood cell counts or urine IL-6 levels; they also showed a higher sensitivity than the results of the urine nitrite test. All patients with significant growth of clinically relevant bacteria had elevated U-HBP levels. The results indicate that rapid analysis of U-HBP can provide helpful guidance in the management of children with suspected UTI.

  9. Stathmin protein level, a potential predictive marker for taxane treatment response in endometrial cancer.

    Directory of Open Access Journals (Sweden)

    Henrica M J Werner

    Full Text Available Stathmin is a prognostic marker in many cancers, including endometrial cancer. Preclinical studies, predominantly in breast cancer, have suggested that stathmin may additionally be a predictive marker for response to paclitaxel. We first evaluated the response to paclitaxel in endometrial cancer cell lines before and after stathmin knock-down. Subsequently we investigated the clinical response to paclitaxel containing chemotherapy in metastatic endometrial cancer in relation to stathmin protein level in tumors. Stathmin level was also determined in metastatic lesions, analyzing changes in biomarker status on disease progression. Knock-down of stathmin improved sensitivity to paclitaxel in endometrial carcinoma cell lines with both naturally higher and lower sensitivity to paclitaxel. In clinical samples, high stathmin level was demonstrated to be associated with poor response to paclitaxel containing chemotherapy and to reduced disease specific survival only in patients treated with such combination. Stathmin level increased significantly from primary to metastatic lesions. This study suggests, supported by both preclinical and clinical data, that stathmin could be a predictive biomarker for response to paclitaxel treatment in endometrial cancer. Re-assessment of stathmin level in metastatic lesions prior to treatment start may be relevant. Also, validation in a randomized clinical trial will be important.

  10. C-reactive protein levels: a prognostic marker for patients with head and neck cancer?

    Science.gov (United States)

    Kruse, Astrid L; Luebbers, Heinz T; Grätz, Klaus W

    2010-08-02

    Recent advances in understanding complex tumor interactions have led to the discovery of an association between inflammation and cancer, in particular for colon and lung cancer, but only a very few have dealt with oral cancer. Therefore, the aim of the current study was to investigate the significance of preoperative C-reactive protein (CRP) levels as a parameter for development of lymph node metastases or recurrence. In 278 patients with oral cancer, preoperative CRP levels were compared with development of recurrence and metastasis. In 27 patients from the normal CRP group, and in 21 patients from the elevated CRP group, local recurrence was observed. Concerning lymph node metastases, 37 patients were in the normal group and 9 patients in the elevated CRP group. No significant correlation could be found between elevated CRP levels and metastasis (p = 0.468) or recurrence (p = 0.137). Our findings do not appear to support a correlation between preoperative CRP levels and development of recurrence or metastases. In further studies, CRP levels in precancerous lesions and in Human Papilloma Virus (HPV) positive patients with oral squamous cell carcinoma (SCC) should be studied.

  11. Effects of Dietary Crude Protein Levels and Cysteamine Supplementation on Protein Synthetic and Degradative Signaling in Skeletal Muscle of Finishing Pigs.

    Directory of Open Access Journals (Sweden)

    Ping Zhou

    Full Text Available Dietary protein levels and cysteamine (CS supplementation can affect growth performance and protein metabolism of pigs. However, the influence of dietary protein intake on the growth response of CS-treated pigs is unclear, and the mechanisms involved in protein metabolism remain unknown. Hence, we investigated the interactions between dietary protein levels and CS supplementation and the effects of dietary crude protein levels and CS supplementation on protein synthetic and degradative signaling in skeletal muscle of finishing pigs. One hundred twenty barrows (65.84 ± 0.61 kg were allocated to a 2 × 2 factorial arrangement with five replicates of six pigs each. The primary variations were dietary crude protein (CP levels (14% or 10% and CS supplemental levels (0 or 700 mg/kg. The low-protein (LP diets (10% CP were supplemented with enough essential amino acids (EAA to meet the NRC AA requirements of pigs and maintain the balanced supply of eight EAA including lysine, methionine, threonine, tryptophan, valine, phenylalanine, isoleucine, and leucine. After 41 days, 10 pigs per treatment were slaughtered. We found that LP diets supplemented with EAA resulted in decreased concentrations of plasma somatostatin (SS (P<0.01 and plasma urea nitrogen (PUN (P<0.001, while dietary protein levels did not affect other traits. However, CS supplementation increased the average daily gain (P<0.001 and lean percentage (P<0.05, and decreased the feed conversion ratio (P<0.05 and back fat (P<0.05. CS supplementation also increased the concentrations of plasma insulin-like growth factor 1 (IGF-1 (P<0.001, and reduced the concentrations of leptin, SS, and PUN (P<0.001. Increased mRNA abundance of Akt1 and IGF-1 signaling (P<0.001 and decreased mRNA abundance of Forkhead Box O (FOXO 4 (P<0.01 and muscle atrophy F-box (P<0.001 were observed in pigs receiving CS. Additionally, CS supplementation increased the protein levels for the phosphorylated mammalian target of

  12. Elevated C-Reactive Protein Levels, Psychological Distress, and Depression in 73 131 Individuals

    DEFF Research Database (Denmark)

    Wium-Andersen, Marie Kim; Ørsted, David Dynnes; Nielsen, Sune Fallgaard

    2013-01-01

    and depression. DESIGN We performed cross-sectional and prospective analyses of CRP levels in 4 clinically relevant categories using data from 2 general population studies. SETTING The Copenhagen General Population and the Copenhagen City Heart studies. PARTICIPANTS We examined 73 131 men and women aged 20......CONTEXT The pathogenesis of depression is not fully understood, but studies suggest that low-grade systemic inflammation contributes to the development of depression. OBJECTIVE To test whether elevated plasma levels of C-reactive protein (CRP) are associated with psychological distress...... to 100 years. MAIN OUTCOME MEASURES We ascertained psychological distress with 2 single-item self-reports and depression using self-reported antidepressant use, register-based prescription of antidepressants, and register-based hospitalization with depression. RESULTS In cross-sectional analyses...