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Sample records for aldh1a2 protein levels

  1. Maternal and zygotic aldh1a2 activity is required for pancreas development in zebrafish.

    PubMed

    Alexa, Kristen; Choe, Seong-Kyu; Hirsch, Nicolas; Etheridge, Letitiah; Laver, Elizabeth; Sagerström, Charles G

    2009-12-11

    We have isolated and characterized a novel zebrafish pancreas mutant. Mutant embryos lack expression of isl1 and sst in the endocrine pancreas, but retain isl1 expression in the CNS. Non-endocrine endodermal gene expression is less affected in the mutant, with varying degrees of residual expression observed for pdx1, carbA, hhex, prox1, sid4, transferrin and ifabp. In addition, mutant embryos display a swollen pericardium and lack fin buds. Genetic mapping revealed a mutation resulting in a glycine to arginine change in the catalytic domain of the aldh1a2 gene, which is required for the production of retinoic acid from vitamin A. Comparison of our mutant (aldh1a2(um22)) to neckless (aldh1a2(i26)), a previously identified aldh1a2 mutant, revealed similarities in residual endodermal gene expression. In contrast, treatment with DEAB (diethylaminobenzaldehyde), a competitive reversible inhibitor of Aldh enzymes, produces a more severe phenotype with complete loss of endodermal gene expression, indicating that a source of Aldh activity persists in both mutants. We find that mRNA from the aldh1a2(um22) mutant allele is inactive, indicating that it represents a null allele. Instead, the residual Aldh activity is likely due to maternal aldh1a2, since we find that translation-blocking, but not splice-blocking, aldh1a2 morpholinos produce a phenotype similar to DEAB treatment. We conclude that Aldh1a2 is the primary Aldh acting during pancreas development and that maternal Aldh1a2 activity persists in aldh1a2(um22) and aldh1a2(i26) mutant embryos.

  2. ALDH1A2 — EDRN Public Portal

    Cancer.gov

    From NCBI Gene: This protein belongs to the aldehyde dehydrogenase family of proteins. The product of this gene is an enzyme that catalyzes the synthesis of retinoic acid (RA) from retinaldehyde. Retinoic acid, the active derivative of vitamin A (retinol), is a hormonal signaling molecule that functions in developing and adult tissues. The studies of a similar mouse gene suggest that this enzyme and the cytochrome CYP26A1, concurrently establish local embryonic retinoic acid levels which facilitate posterior organ development and prevent spina bifida. Four transcript variants encoding distinct isoforms have been identified for this gene. [provided by RefSeq, May 2011

  3. Zebrafish foxc1a plays a crucial role in early somitogenesis by restricting the expression of aldh1a2 directly.

    PubMed

    Li, Jingyun; Yue, Yunyun; Dong, Xiaohua; Jia, Wenshuang; Li, Kui; Liang, Dong; Dong, Zhangji; Wang, Xiaoxiao; Nan, Xiaoxi; Zhang, Qinxin; Zhao, Qingshun

    2015-04-17

    Foxc1a is a member of the forkhead transcription factors. It plays an essential role in zebrafish somitogenesis. However, little is known about the molecular mechanisms underlying its controlling somitogenesis. To uncover how foxc1a regulates zebrafish somitogenesis, we generated foxc1a knock-out zebrafish using TALEN (transcription activator-like effector nuclease) technology. The foxc1a null embryos exhibited defective somites at early development. Analyses on the expressions of the key genes that control processes of somitogenesis revealed that foxc1a controlled early somitogenesis by regulating the expression of myod1. In the somites of foxc1a knock-out embryos, expressions of fgf8a and deltaC were abolished, whereas the expression of aldh1a2 (responsible for providing retinoic acid signaling) was significantly increased. Once the increased retinoic acid level in the foxc1a null embryos was reduced by knocking down aldh1a2, the reduced expression of myod1 was partially rescued by resuming expressions of fgf8a and deltaC in the somites of the mutant embryos. Moreover, a chromatin immunoprecipitation assay on zebrafish embryos revealed that Foxc1a bound aldh1a2 promoter directly. On the other hand, neither knocking down fgf8a nor inhibiting Notch signaling affected the expression of aldh1a2, although knocking down fgf8a reduced expression of deltaC in the somites of zebrafish embryos at early somitogenesis and vice versa. Taken together, our results demonstrate that foxc1a plays an essential role in early somitogenesis by controlling Fgf and Notch signaling through restricting the expression of aldh1a2 in paraxial mesoderm directly.

  4. ALDH1A2 (RALDH2) genetic variation in human congenital heart disease

    PubMed Central

    2009-01-01

    Background Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus. Methods One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay. Results We describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls. Conclusion In summary, our screen indicates that ALDH1A2 genetic

  5. Beta 1-integrin ligation and TLR ligation enhance GM-CSF–induced ALDH1A2 expression in dendritic cells, but differentially regulate their anti-inflammatory properties

    PubMed Central

    Yokota-Nakatsuma, Aya; Ohoka, Yoshiharu; Takeuchi, Hajime; Song, Si-Young; Iwata, Makoto

    2016-01-01

    Retinoic acid (RA)–producing CD103+ mature dendritic cells (DCs) in mesenteric lymph nodes (MLNs) play crucial roles in gut immunity. GM-CSF and RA contribute to the expression of the RA-producing enzyme ALDH1A2. However, additional signals appeared to be required for inducing ALDH1A2high mature DCs from immature DCs. We found here that TLR ligands (Ls) and immobilized E-cadherin could provide such signals in FLT3-L–generated bone marrow (BM)–derived DCs after treatment with GM-CSF and the RA receptor agonist Am80. The TLR-L-treated DCs produced proinflammatory cytokines unlike normal ALDH1A2high MLN-DCs, whereas the E-cadherin-treated DCs did not. Immobilized VCAM-1 and semaphorin 7 A exerted effects similar to those of E-cadherin. Soluble anti-integrin β1 antibodies or inhibitors of integrin signaling molecules suppressed the effects of these immobilized proteins, whereas immobilized anti-integrin β1 antibodies enhanced the GM-CSF/Am80-induced ALDH1A2 expression without inducing proinflammatory cytokines. Sequential stimulation of splenic pre-DCs with GM-CSF/Am80 and immobilized E-cadherin or anti-integrin β1 antibody also induced differentiation to mature DCs with high ALDH activity. The E-cadherin-treated BM-DCs induced gut-tropic Foxp3+ T cells and alleviated DSS–induced colitis, whereas the TLR-L-treated DCs aggravated DSS–induced colitis. The results suggest that integrin β1-mediated signals contribute to the differentiation and maturation of RA-producing anti-inflammatory DCs. PMID:27897208

  6. Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus)

    PubMed Central

    Feng, Ruijuan; Fang, Lingling; Cheng, Yunying; He, Xue; Jiang, Wentao; Dong, Ranran; Shi, Hongjuan; Jiang, Dongneng; Sun, Lina; Wang, Deshou

    2015-01-01

    Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts. PMID:25976364

  7. Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus).

    PubMed

    Feng, Ruijuan; Fang, Lingling; Cheng, Yunying; He, Xue; Jiang, Wentao; Dong, Ranran; Shi, Hongjuan; Jiang, Dongneng; Sun, Lina; Wang, Deshou

    2015-05-15

    Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts.

  8. Retinol status and expression of retinol-related proteins in methionine-choline deficient rats.

    PubMed

    Miyazaki, Hiroshi; Takitani, Kimitaka; Koh, Maki; Inoue, Akiko; Kishi, Kanta; Tamai, Hiroshi

    2014-01-01

    Retinol and its derivative, retinoic acid, have pleiotropic functions including vision, immunity, hematopoiesis, reproduction, cell differentiation/growth, and development. Non-alcoholic fatty liver disease (NAFLD) is one of the most common diseases in developed countries and encompasses a broad spectrum of forms, ranging from steatosis to steatohepatitis, which develops further to cirrhosis. Retinol status has an important role in liver homeostasis. The purpose of this study was to evaluate the retinol status and expression of retinol-related proteins, including enzymes and binding proteins, in methionine-choline deficient (MCD) rats as a model of NAFLD. We examined retinol levels in the plasma and liver and gene expression for β-carotene 15,15'-monooxygenase (BCMO), lecithIn: retinol acyltransferase (LRAT), aldehyde dehydrogenase 1A1 (ALDH1A1), ALDH1A2, and cellular retinol binding protein (CRBP)-I in MCD rats. The plasma retinol levels in MCD rats were lower than those in the controls, whereas hepatic retinol levels in MCD rats were higher. BCMO expression in the intestine and liver in MCD rats was lower, whereas that in the testes and the kidneys was higher than in control rats. Expression of LRAT, CRBP-I, ALDH1A1, and ALDH1A2 in the liver of MCD rats was also higher. Altered expression of retinol-related proteins may affect retinol status in NAFLD.

  9. Can protein levels be economically increased?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One result from the 2010 hard red winter wheat harvest was an increase of discussions on protein values across the southern great plains. The crop garnered relatively low protein values for several reasons, many of which were directly related to the weather patterns and environmental conditions. T...

  10. Ethanol Disrupts Chondrification of the Neurocranial Cartilages in Medaka Embryos without Affecting Aldehyde Dehydrogenase 1A2 (Aldh1A2) Promoter Methylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Medaka (Oryzias latipes) embryos at different developmental stages were exposed to ethanol for 48 h, then allowed to hatch. Teratogenic effects were evaluated in hatchlings after examining chondrocranial cartilage deformities. Ethanol disrupted cartilage development in medaka in a dose and developme...

  11. Dietary protein source and level alters growth in neon tetras.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nutritional studies for aquarium fish like the neon tetra are sparse in comparison with those for food fish. To determine the optimum dietary protein level and source for growth of neon tetras, diets were formulated to contain 25, 35, 45 and 55% dietary protein from either marine animal protein or ...

  12. Dietary protein level and performance of growing Baladi kids

    PubMed Central

    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 (P<0.05) the total live weight gain, average daily gain and feed conversion ratio when compared with the control and T3 groups. Moreover, treatment, time and time × treatment caused a significant change on Co concentration in blood serum with higher value at the end of the experiment. Treatments had a significant effect (P<0.05) on blood serum cholesterol and protein levels. Undegradable methionine supplementation (T3) significantly increased longissimus dorsi weight, fat thickness and omental fat%. In conclusion, feeding Baladi kids 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. PMID:27175130

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

    PubMed

    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 (P<0.05) the total live weight gain, average daily gain and feed conversion ratio when compared with the control and T3 groups. Moreover, treatment, time and time × treatment caused a significant change on Co concentration in blood serum with higher value at the end of the experiment. Treatments had a significant effect (P<0.05) on blood serum cholesterol and protein levels. Undegradable methionine supplementation (T3) significantly increased longissimus dorsi weight, fat thickness and omental fat%. In conclusion, feeding Baladi kids 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.

  14. Soy protein isolate molecular level contributions to bulk adhesive properties

    NASA Astrophysics Data System (ADS)

    Shera, Jeanne Norton

    Increasing environmental awareness and the recognized health hazards of formaldehyde-based resins has prompted a strong demand for environmentally-responsible adhesives for wood composites. Soy protein-based adhesives have been shown to be commercially viable with 90-day shelf stability and composite physical properties comparable to those of commercial formaldehyde-based particleboards. The main research focus is to isolate and characterize the molecular level features in soy protein isolate responsible for providing mechanical properties, storage stability, and water resistance during adhesive formulation, processing, and wood composite fabrication. Commercial composite board will be reviewed to enhance our understanding of the individual components and processes required for particleboard production. The levels of protein structure will be defined and an overview of current bio-based technology will be presented. In the process, the logic for utilizing soy protein as a sole binder in the adhesive will be reinforced. Variables such as adhesive components, pH, divalent ions, blend aging, protein molecular weight, formulation solids content, and soy protein functionalization will relate the bulk properties of soy protein adhesives to the molecular configuration of the soybean protein. This work has demonstrated that when intermolecular beta-sheet interactions and protein long-range order is disrupted, viscosity and mechanical properties decrease. Storage stability can be maintained through the stabilization of intermolecular beta-sheet interactions. When molecular weight is reduced through enzymatic digestion, long-range order is disrupted and viscosity and mechanical properties decrease accordingly. Processibility and physical properties must be balanced to increase solids while maintaining low viscosity, desirable mechanical properties, and adequate storage stability. The structure of the soybean protein must be related to the particleboard bulk mechanical

  15. Plasma amino acid response to graded levels of escape protein.

    PubMed

    Gibb, D J; Klopfenstein, T J; Britton, R A; Lewis, A J

    1992-09-01

    A trial was conducted to examine the potential of using plasma amino acid responses to graded levels of escape protein to determine limiting amino acids in cattle. Growing calves (n = 120; mean BW = 220 +/- 21 kg) were fed a basal diet of corncob:sorghum silage (61:39) and were individually supplemented with distillers' dried grains (DDG), heat-damaged DDG (H-DDG), feather meal (FTH), or urea. The urea supplement was mixed with DDG and H-DDG to allow 0, 20, 35, 50, 65, or 80% of the supplemental CP to come from distillers' protein and maintain an 11.5% CP diet. Urea supplement was mixed with FTH to allow 0, 22, 39, 56, 73, or 90% of the supplemental CP to come from FTH. Dietary CP ranged from 11.5% at the 0% level to 17.3% at the 90% level. Plasma concentration of most essential plasma amino acids responded (P less than .10) linearly and(or) quadratically to increased escape protein. The broken-line response of plasma methionine at low DDG intake suggested that methionine was limiting at low levels of escape protein. An initial decrease followed by a plateau fit by a broken line indicated that histidine became limiting in FTH diets, and lysine eventually became limiting for DDG, H-DDG, and FTH diets before maximum BW gain was reached. Results indicate that plasma amino acid responses may identify amino acids that become limiting with increasing escape protein.

  16. Smoking, COPD and 3-Nitrotyrosine Levels of Plasma Proteins

    SciTech Connect

    Jin, Hongjun; Webb-Robertson, Bobbie-Jo M.; Peterson, Elena S.; Tan, Ruimin; Bigelow, Diana J.; Scholand, Mary Beth; Hoidal, John R.; Pounds, Joel G.; Zangar, Richard C.

    2011-09-01

    BACKGROUND: Nitric oxide is a physiologically regulator of endothelial function and hemodynamics. Oxidized products of nitric oxide can form nitrotyrosine, which is a marker of nitrative stress. Cigarette smoking decreases exhaled nitric oxide, and the underlying mechanism may be important in the cardiovascular toxicity of cigarette smoke, although it is not clear if this effect results from decreased nitric oxide production or oxidation of nitric oxide to reactive, nitrating, species. These processes would be expected to have opposite effects on nitrotyrosine levels, a marker of nitrative stress. OBJECTIVE: In this study, we determine the effects of smoking and chronic obstructive pulmonary disease (COPD) on circulating levels of nitrotyrosine, and thereby gain insight into the processes regulating nitrotyrosine formation. METHODS: A custom antibody microarray platform was used to analyze the levels of 3-nitrotyrosine modifications on 24 proteins in plasma. Plasma samples from 458 individuals were analyzed. RESULTS: Nitrotyrosine levels in circulating proteins were uniformly reduced in smokers but increased in COPD patients. We also observed a persistent suppression of nitrotyrosine in former smokers. CONCLUSIONS: Smoking broadly suppresses the levels of 3-nitrotyrosine in plasma proteins, suggesting that cigarette smoke suppresses endothelial nitric oxide production. In contrast, the increase in nitrotyrosine levels in COPD patients most likely results from inflammatory processes. This study provides the first evidence that smoking has irreversible effects on endothelial production of nitric oxide, and provides insight into how smoking could induce a loss of elasticity in the vasculature and a long-term increase in the risk of cardiovascular disease.

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

    USGS Publications Warehouse

    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.

  18. Specific serum protein levels in women using intrauterine contraceptive device.

    PubMed

    Wiedermann, D; Kríz, J; Cídl, K

    1980-01-01

    The report is concerned with the levels of 17 specific serum proteins in 46 women using plastic nonmedicated intrauterine contraceptive device (IUCD) Dana-Super. Blood samplings were carried out three times: just before the IUCD introduction, 30 and 54 weeks after the insertion of IUCD. The following proteins except haptoglobin were quantitatively determined by radial immunodiffusion: prealbumin, albumin, orosomucoid, alpha 1-antitrypsin, ceruloplasmin, alpha 2HS-glycoprotein, alpha 2-macroglobulin, hemopexin, C3-component, transferrin, beta 2-glycoprotein I, C-reactive protein and immunoglobulins IgG, IgA, IgM and IgD. Moderately increased values were found for alpha 2HS-glycoprotein and beta 2-glycoprotein I in sera taken 30 weeks after the insertion of IUCD. AT the same time the augmentation of alpha 1-antitrypsin was established. This might be evoked by the raised protease activity in biological fluids of genital region. The raise in consequence of IUCD application of transferrin and the decrease of haptoglobin at the first postinsertion examination and the decrease of hemopexin and albumin at the second may be associated with higher menstrual bleeding followed by iron deficiency. All other proteins as well as the acute phase proteins showed only minor if any differences as compared with the corresponding start values. Similarly, there is no evidence of a systemic immunoglobulin response to IUCD use.

  19. Effect of Crude Protein Levels in Concentrate and Concentrate Levels in Diet on In vitro Fermentation.

    PubMed

    Van Dung, Dinh; Shang, Weiwei; Yao, Wen

    2014-06-01

    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.

  20. Autophagy supports survival and phototransduction protein levels in rod photoreceptors

    PubMed Central

    Zhou, Z; Doggett, T A; Sene, A; Apte, R S; Ferguson, T A

    2015-01-01

    Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod

  1. Importance of ALDH1A enzymes in determining human testicular retinoic acid concentrations

    PubMed Central

    Arnold, Samuel L.; Kent, Travis; Hogarth, Cathryn A.; Schlatt, Stefan; Prasad, Bhagwat; Haenisch, Michael; Walsh, Thomas; Muller, Charles H.; Griswold, Michael D.; Amory, John K.; Isoherranen, Nina

    2015-01-01

    Retinoic acid (RA), the active metabolite of vitamin A, is required for spermatogenesis and many other biological processes. RA formation requires irreversible oxidation of retinal to RA by aldehyde dehydrogenase enzymes of the 1A family (ALDH1A). While ALDH1A1, ALDH1A2, and ALDH1A3 all form RA, the expression pattern and relative contribution of these enzymes to RA formation in the testis is unknown. In this study, novel methods to measure ALDH1A protein levels and intrinsic RA formation were used to accurately predict RA formation velocities in individual human testis samples and an association between RA formation and intratesticular RA concentrations was observed. The distinct localization of ALDH1A in the testis suggests a specific role for each enzyme in controlling RA formation. ALDH1A1 was found in Sertoli cells, while only ALDH1A2 was found in spermatogonia, spermatids, and spermatocytes. In the absence of cellular retinol binding protein (CRBP)1, ALDH1A1 was predicted to be the main contributor to intratesticular RA formation, but when CRBP1 was present, ALDH1A2 was predicted to be equally important in RA formation as ALDH1A1. This study provides a comprehensive novel methodology to evaluate RA homeostasis in human tissues and provides insight to how the individual ALDH1A enzymes mediate RA concentrations in specific cell types. PMID:25502770

  2. Ascertaining effects of nanoscale polymeric interfaces on competitive protein adsorption at the individual protein level

    NASA Astrophysics Data System (ADS)

    Song, Sheng; Xie, Tian; Ravensbergen, Kristina; Hahm, Jong-In

    2016-02-01

    With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those occurring in a competitive adsorption environment. Complex sequences of adhesion events in competitive adsorption involving multicomponent protein systems have been extensively investigated, but our understanding is still limited primarily to macroscopic adhesion onto chemically simple surfaces. We examine the competitive adsorption behavior from a binary protein mixture containing bovine serum albumin and fibrinogen at the single protein level. We subsequently evaluate a series of adsorption and displacement processes occurring on both the macroscopic homopolymer and nanoscopic diblock copolymer surfaces, while systematically varying the protein concentration and incubation time. We identify the similarities and dissimilarities in competitive protein adsorption behavior between the two polymeric surfaces, the former presenting chemical uniformity at macroscale versus the latter exhibiting periodic nanointerfaces of chemically alternating polymeric segments. We then present our novel experimental finding of a large increase in the nanointerface-engaged residence time of the initially bound proteins and further explain the origin of this phenomenon manifested on nanoscale diblock copolymer surfaces. The outcomes of this study may provide timely insight into nanoscale competitive protein adsorption that is much needed in designing bioimplant and tissue engineering materials. In addition, the fundamental understanding gained from this study can be beneficial for the development of highly miniaturized biodevices and biomaterials fabricated by using nanoscale polymeric materials and interfaces.With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those

  3. Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

    PubMed Central

    Chen, Ruidong; Zhao, Xue; Wang, Yongxiang; Xie, Youhua; Liu, Jing

    2017-01-01

    The apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) family proteins bind RNA and single-stranded DNA, and create C-to-U base modifications through cytidine deaminase activity. APOBEC3G restricts human immunodeficiency virus 1 (HIV-1) infection by creating hypermutations in proviral DNA, while HIV-1-encoded vif protein antagonizes such restriction by targeting APOBEC3G for degradation. APOBEC3G also inhibits hepatitis B virus (HBV): APOBEC3G co-expression inhibits HBV replication and evidences exist indicating APOBEC3G-mediated HBV hypermutations in patients. HBV encodes a small non-structural X protein (HBx) with a recognized activating effect on HBV life cycle. In this work, we report the discovery that HBx selectively and dose-dependently decreases the protein level of co-expressed APOBEC3G in transfected Huh-7 cells. The effect was shown to take place post-translationally, but does not rely on protein degradation via proteasome or lysosome. Further work demonstrated that intracellular APOBEC3G is normally exported via exosome secretion and inhibition of exosome biogenesis causes retention of intracellular APOBEC3G. Finally, HBx co-expression specifically enhanced externalization of APOBEC3G via exosomes, resulting in decrease of intracellular APOBEC3G protein level. These data suggest the possibility that in addition to other mechanisms, HBx-mediated activation of HBV might also involve antagonizing of intracellular restriction factor APOBEC3G through promotion of its export. PMID:28098260

  4. Effects of soy protein and calcium levels on mineral bioaccessibility and protein digestibility from enteral formulas.

    PubMed

    Galán, María Gimena; Drago, Silvina Rosa

    2014-09-01

    Enteral formulas (EF) are complex food systems which have all the nutrients in their matrix for the complete human nourishment. However, there are components in EF which can interact with minerals, reducing their absorption, and thereof the EF nutritional quality. The effect of soy protein (SP) and Ca content on Fe, Zn, and Ca bioaccessibility and protein digestibility (%DP) was assessed using a response surface design in EF. Tested SP levels were 2.5-5.0 g/100 mL of total protein. Ca levels were adjusted with Ca citrate within a range between 50 and 100 mg/100 mL. SP content negatively influenced %DP and Fe, Zn and Ca bioaccessibility. As SP content increased, mineral bioaccessibility and %DP decreased, probably due to the increased levels of phytic acid and trypsin inhibitors from SP. Ca content only affected %DCa, which had a direct relationship with Ca levels, while did not affect Fe and Zn bioaccessibility or %DP. Since Ca citrate did not impair Fe and Zn bioaccessibility, it could be an appropriate Ca source for EF fortification.

  5. Honey bee protein atlas at organ-level resolution

    PubMed Central

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

    2013-01-01

    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. PMID:23878156

  6. Imaging proteins at the single-molecule level

    PubMed Central

    Longchamp, Jean-Nicolas; Rauschenbach, Stephan; Abb, Sabine; Escher, Conrad; Latychevskaia, Tatiana; Kern, Klaus; Fink, Hans-Werner

    2017-01-01

    Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics, and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical- and conformational-specific selection and gentle deposition. Low-energy electrons do not induce radiation damage, which enables acquiring subnanometer resolution images of individual proteins (cytochrome C and BSA) as well as of protein complexes (hemoglobin), which are not the result of an averaging process. PMID:28087691

  7. Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants.

    PubMed

    Conley, Andrew J; Joensuu, Jussi J; Richman, Alex; Menassa, Rima

    2011-05-01

    For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.

  8. Microsecond protein dynamics observed at the single-molecule level

    PubMed Central

    Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

    2015-01-01

    How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape. PMID:26151767

  9. Development, characterization, and optimization of protein level in date bars using response surface methodology.

    PubMed

    Nadeem, Muhammad; Salim-ur-Rehman; Muhammad Anjum, Faqir; Murtaza, Mian Anjum; Mueen-ud-Din, Ghulam

    2012-01-01

    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.

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

    PubMed Central

    Nadeem, Muhammad; Salim-ur-Rehman; Muhammad Anjum, Faqir; Murtaza, Mian Anjum; Mueen-ud-Din, Ghulam

    2012-01-01

    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. PMID:22792044

  11. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    NASA Astrophysics Data System (ADS)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  12. Protein Z variants associated with protein Z plasma levels and with risk of idiopathic recurrent miscarriage.

    PubMed

    Al-Shaikh, Fatima S; Sater, Mai S; Finan, Ramzi R; Racoubian, Eddie; Abu-Hijleh, Tala M; Mustafa, Fekria E; Almawi, Wassim Y

    2013-09-01

    Protein Z (PZ) deficiency due to anti-PZ autoantibodies and/or mutations in PZgene was linked with adverse pregnancy outcomes, including idiopathic recurrent miscarriage (IRM). We investigated the association of rs3024718, rs3024719, rs3024731, rs3024778, rs3024772, and rs3024735 (G79A) PZ variants and changes in PZ levels in 287 women with IRM, and 308 control women. Of the 6 single nucleotide polymorphisms (SNPs) analyzed, higher minor allele frequency of rs3024735 (G79A) and rs3024731 were seen in IRM cases than in control women. Significantly higher frequencies of rs3024735/G79A G/A and A/A (P< .001), rs3024719 G/A (P= .009), and rs3024731 A/A (P = .012), but not rs3024718 (P= .12), rs3024778 (P = .76), or rs3024772 (P= .27) genotype carriers were seen between IRM cases versus control women, respectively, and was linked with reduced PZ levels. Six-locus (rs3024718/rs3024719/rs3024778/rs3024731/rs3024735/rs3024772) PZhaplotypes analysis demonstrated increased frequency of GAGAAG and AGGTAG and reduced frequency of AGGTGC haplotypes in IRM cases, thereby conferring disease susceptibility and protective nature to these haplotypes, respectively. These results demonstrate that specific PZSNPs and haplotypes are significantly associated with IRM.

  13. Association of protein S p.Pro667Pro dimorphism with plasma protein S levels in normal individuals and patients with inherited protein S deficiency.

    PubMed

    Castaman, G; Biguzzi, E; Razzari, C; Tosetto, A; Fontana, G; Asti, D; Brancaccio, V; Castori, D; Lane, D A; Faioni, E M

    2007-01-01

    A dimorphism in PROS1 gene (c.A2,001G, p.Pro667Pro) has been associated with significantly reduced levels of both free and total protein S in carriers of the GG genotype. It is not known how the GG genotype could influence PS levels in normals, whether it could influence the levels of protein S in carriers of mutations in PROS1 gene and whether this genotype acts as an isolated or additive risk factor for venous thrombosis. With this as background, we evaluated the association of p.Pro667Pro dimorphism with free and total protein S centrally measured in a panel of 119 normal controls, 222 individuals with low protein S and 137 individuals with normal PS levels belonging to 76 families with protein S deficiency enrolled in the ProSIT study. Transient expression of recombinant wild type protein S and p.Pro667Pro protein S was performed to evaluate the role of the A to G transition at position 2001 in vitro. The p.Pro667Pro polymorphism was also expressed together with a p.Glu67Ala variant to assess a possible influence on protein S levels in protein S deficient subjects. Free and total protein S levels were significantly lower in normal women. In normal women only was the GG genotype associated with significantly lower free protein S levels in comparison to AA and AG genotypes (P=0.032). No significant influence of GG genotype was observed in patients, either with known mutations or with low protein S levels. These data were confirmed by in vitro transient expression, showing no difference in secretion levels of the p.Pro667Pro variant (even in association with the p.Glu67Ala mutation), compared to the wild type protein S. The genotype in itself was neither a significant risk factor for venous thrombosis nor a risk modifier in patients with known mutations.

  14. Methods to alter levels of a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-10-17

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  15. Multifactorial level of extremostability of proteins: can they be exploited for protein engineering?

    PubMed

    Chakravorty, Debamitra; Khan, Mohd Faheem; Patra, Sanjukta

    2017-03-10

    Research on extremostable proteins has seen immense growth in the past decade owing to their industrial importance. Basic research of attributes related to extreme-stability requires further exploration. Modern mechanistic approaches to engineer such proteins in vitro will have more impact in industrial biotechnology economy. Developing a priori knowledge about the mechanism behind extreme-stability will nurture better understanding of pathways leading to protein molecular evolution and folding. This review is a vivid compilation about all classes of extremostable proteins and the attributes that lead to myriad of adaptations divulged after an extensive study of 6495 articles belonging to extremostable proteins. Along with detailing on the rationale behind extreme-stability of proteins, emphasis has been put on modern approaches that have been utilized to render proteins extremostable by protein engineering. It was understood that each protein shows different approaches to extreme-stability governed by minute differences in their biophysical properties and the milieu in which they exist. Any general rule has not yet been drawn regarding adaptive mechanisms in extreme environments. This review was further instrumental to understand the drawback of the available 14 stabilizing mutation prediction algorithms. Thus, this review lays the foundation to further explore the biophysical pleiotropy of extreme-stable proteins to deduce a global prediction model for predicting the effect of mutations on protein stability.

  16. Protein metabolism in growing pigs fed corn or cassava peel based diets containing graded protein levels.

    PubMed

    Tewe, O O

    1985-05-01

    Sixty-four Large White cross Landrace weanling pigs were randomly allotted to eight treatments in a two by four factorial arrangement. The two dietary variables were cassava peel (0 and 40 per cent) and crude protein (20, 15, 10 and 5 per cent). Total serum protein concentration was significantly (P less than 0.01) reduced by protein deficiency and by its interaction with cassava peel. The multiple coefficient of determination (R2) showed that protein intake was the primary factor determining changes in serum protein. R2 values for cyanide intake (independent variable) on serum protein (dependent variable) increased from day 30 to 90 of the trial. Serum urea was increased on the 5 per cent protein diets on days 60 and 90 of the trial. The R2 values for cyanide and protein intake on serum urea concentration increased from day 30 to day 90 of the trial. Serum creatinine increased (P less than 0.05) on the 5 per cent protein diet on day 90 of the trial. The R2 value for the effects of protein intake on serum creatinine was higher than for cyanide intake on days 30 and 90. The results confirm the progressive and pronounced effects of long term cyanide intake on serum nitrogenous metabolites in pigs consuming between 110 and 120 ppm hydrocyanic acid, especially in diets containing 10 per cent or less protein.

  17. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis1

    PubMed Central

    Pesaresi, Paolo; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Rothbart, Maxi; Hedtke, Boris

    2016-01-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes. PMID:26823545

  18. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis.

    PubMed

    Tadini, Luca; Pesaresi, Paolo; Kleine, Tatjana; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Pribil, Mathias; Rothbart, Maxi; Hedtke, Boris; Grimm, Bernhard; Leister, Dario

    2016-03-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes.

  19. The level of Yop proteins secreted by Yersinia enterocolitica is changed in maltose mutants.

    PubMed

    Brzostek, K; Raczkowska, A

    2001-10-16

    Enteropathogenic Yersinia enterocolitica strains express a set of plasmid-encoded proteins called Yops, involved in pathogenicity. We studied the influence of the maltose system on the production of Yop proteins and found that the level of Yop proteins of Y. enterocolitica O:9 was reduced in the presence of maltose. Transposon insertion mutants impaired with the maltose transport activity showed a decreased level in the production of Yop proteins. The transcription of the yopH gene for YopH phosphatase in these maltose mutants was unchanged and revealed a maltose mutation impaired in the secretion of Yop proteins instead of their expression.

  20. The Hsp90-Dependent Proteome Is Conserved and Enriched for Hub Proteins with High Levels of Protein–Protein Connectivity

    PubMed Central

    Swamy, Krishna B.S.; Yu, Jau-Song; Schuyler, Scott C.; Leu, Jun-Yi

    2014-01-01

    Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic and nongenetic variation within a population. Despite information about Hsp90 protein–protein interactions, a global view of the Hsp90-regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins on Hsp90, we used the “stable isotope labeling by amino acids in cell culture” method coupled with mass spectrometry to quantify around 4,000 proteins in low-Hsp90 cells. We observed that 904 proteins changed in their abundance by more than 1.5-fold. When compared with the transcriptome of the same population of cells, two-thirds of the misregulated proteins were observed to be affected posttranscriptionally, of which the majority were downregulated. Further analyses indicated that the downregulated proteins are highly conserved and assume central roles in cellular networks with a high number of protein interacting partners, suggesting that Hsp90 buffers genetic and nongenetic variation through regulating protein network hubs. The downregulated proteins were enriched for essential proteins previously not known to be Hsp90-dependent. Finally, we observed that downregulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response, respectively, providing a direct link between observed proteome regulation and cellular phenotypes. PMID:25316598

  1. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.

    PubMed

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-04-21

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise.

  2. Impact of genetic variation on synaptic protein levels in genetically diverse mice.

    PubMed

    Loos, Maarten; Li, Ka Wan; van der Schors, Roel; Gouwenberg, Yvonne; van der Loo, Rolinka; Williams, Robert W; Smit, August B; Spijker, Sabine

    2016-04-01

    The relative abundance of synaptic proteins shapes protein complex formation and is essential for synapse function and behavioral fitness. Here, we have used a panel of highly diverse inbred strains of mice-NOD/LtJ, A/J, 129S1/SvImJ, FVB/NJ, C57BL/6J, WSB/EiJ, PWK/PhJ, and CAST/EiJ-to quantify the effects of genetic variation on the synaptic proteome between strains. Using iTRAQ-based quantitative proteome analyses, we detected significant differences in ∼20% of 400 core synaptic proteins. Surprisingly, the differentially abundant proteins showed a modest range of variation across strains, averaging about 1.3-fold. Analysis of protein abundance covariation across the eight strains identified known protein-protein relations (proteins of Arp2/3 complex), as well as novel relations (e.g. Dlg family, Fscn1). Moreover, covariation of synaptic proteins was substantially tighter (∼fourfold more dense than chance level) than corresponding networks of synaptic transcripts (∼twofold more dense than chance). The tight stoichiometry and coherent synaptic protein covariation networks suggest more intense evolutionary selection at this level of molecular organization. In conclusion, genetic diversity in the mouse genome differentially affects the transcriptome and proteome, and only partially penetrates the synaptic proteome. Protein abundance correlation analyses in genetically divergent strains can complement protein-protein interaction network analyses, to provide insight into protein interactomes.

  3. Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels

    PubMed Central

    2013-01-01

    Background In Escherichia coli many heterologous proteins are produced in the periplasm. To direct these proteins to the periplasm, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec-translocon. For poorly understood reasons, the production of heterologous secretory proteins is often toxic to the cell thereby limiting yields. To gain insight into the mechanism(s) that underlie this toxicity we produced two secretory heterologous proteins, super folder green fluorescent protein and a single-chain variable antibody fragment, in the Lemo21(DE3) strain. In this strain, the expression intensity of the gene encoding the target protein can be precisely controlled. Results Both SFGFP and the single-chain variable antibody fragment were equipped with a DsbA-derived signal sequence. Producing these proteins following different gene expression levels in Lemo21(DE3) allowed us to identify the optimal expression level for each target gene. Too high gene expression levels resulted in saturation of the Sec-translocon capacity as shown by hampered translocation of endogenous secretory proteins and a protein misfolding/aggregation problem in the cytoplasm. At the optimal gene expression levels, the negative effects of the production of the heterologous secretory proteins were minimized and yields in the periplasm were optimized. Conclusions Saturating the Sec-translocon capacity can be a major bottleneck hampering heterologous protein production in the periplasm. This bottleneck can be alleviated by harmonizing expression levels of the genes encoding the heterologous secretory proteins with the Sec-translocon capacity. Mechanistic insight into the production of proteins in the periplasm is key to optimizing yields in this compartment. PMID:23497240

  4. DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein.

    PubMed

    Ferreira, Daniela M; Miyaji, Eliane N; Oliveira, Maria Leonor S; Darrieux, Michelle; Arêas, Ana Paula M; Ho, Paulo L; Leite, Luciana C C

    2006-04-01

    Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.

  5. Effects of the SpoVT Regulatory Protein on the Germination and Germination Protein Levels of Spores of Bacillus subtilis

    PubMed Central

    Ramirez-Peralta, Arturo; Stewart, Kerry-Ann V.; Thomas, Stacy K.; Setlow, Barbara; Chen, Zhan; Li, Yong-qing

    2012-01-01

    Bacillus subtilis isolates lacking the SpoVT protein, which regulates gene expression in developing forespores, gave spores that released their dipicolinic acid (DPA) via germinant receptor (GR)-dependent germination more rapidly than wild-type spores. Non-GR-dependent germination via dodecylamine was more rapid with spoVT spores, but germination via Ca-DPA was slower. The effects of a spoVT mutation on spore germination were seen with spores made in rich and poor media, and levels of SpoVT-LacZ were elevated 2-fold in poor-medium spores; however, elevated SpoVT levels were not the only cause of the slower GR-dependent germination of poor-medium spores. The spoVT spores had ≥5-fold higher GerA GR levels, ∼2-fold elevated GerB GR levels, wild-type levels of a GerK GR subunit and the GerD protein required for normal GR-dependent germination, ∼2.5-fold lower levels of the SpoVAD protein involved in DPA release in spore germination, and 30% lower levels of DNA protective α/β-type small, acid-soluble spore proteins. With one exception, the effects on protein levels in spoVT spores are consistent with the effects of SpoVT on forespore transcription. The spoVT spores were also more sensitive to UV radiation and outgrew slowly. While spoVT spores' elevated GR levels were consistent with their more rapid GR-dependent germination, detailed analysis of the results suggested that there is another gene product crucial for GR-dependent spore germination that is upregulated in the absence of SpoVT. Overall, these results indicate that SpoVT levels during spore formation have a major impact on the germination and the resistance of the resultant spores. PMID:22522895

  6. Interactive Effects of Indigestible Carbohydrates, Protein Type, and Protein Level on Biomarkers of Large Intestine Health in Rats

    PubMed Central

    Taciak, Marcin; Barszcz, Marcin; Tuśnio, Anna; Pastuszewska, Barbara

    2015-01-01

    The effects of indigestible carbohydrates, protein type, and protein level on large intestine health were examined in rats. For 21 days, 12 groups of six 12-week-old male Wistar rats were fed diets with casein (CAS), or potato protein concentrate (PPC), providing 14% (lower protein level; LP), or 20% (higher protein level; HP) protein, and containing cellulose, resistant potato starch, or pectin. Fermentation end-products, pH, and β-glucuronidase levels in cecal digesta, and ammonia levels in colonic digesta were determined. Cecal digesta, tissue weights, cecal and colon morphology, and colonocyte DNA damage were also analyzed. Digesta pH was lower, whereas relative mass of cecal tissue and digesta were higher in rats fed pectin diets than in those fed cellulose. Cecal parameters were greater in rats fed PPC and HP diets than in those fed CAS and LP diets, respectively. Short-chain fatty acid (SCFA) concentrations were unaffected by protein or carbohydrate type. Total SCFA, acetic acid, and propionic acid concentrations were greater in rats fed LP diets than in those fed HP. Cecal pool of isobutyric and isovaleric acids was greater in rats fed PPC than in those fed CAS diets. PPC diets decreased phenol concentration and increased ammonia concentration in cecal and colonic digesta, respectively. Cecal crypt depth was greater in rats fed PPC and HP diets, and was unaffected by carbohydrates; whereas colonic crypt depth was greater in rats fed cellulose. Myenteron thickness in the cecum was unaffected by nutrition, but was greater in the colon of rats fed cellulose. Colonocyte DNA damage was greater in rats fed LP diets than in those fed HP diets, and was unaffected by carbohydrate or protein type. It was found that nutritional factors decreasing cecal digesta weight contribute to greater phenol production, increased DNA damage, and reduced ammonia concentration in the colon. PMID:26536028

  7. Calcium, phosphorus and protein levels as factors in the distribution of the pheasant

    USGS Publications Warehouse

    Dale, F.H.; DeWitt, J.B.

    1958-01-01

    Summary of work on pheasant nutrition conducted since 1949 at the Patuxent Research Refuge. Pheasant chicks fed experimental diets failed to develop normally on protein levels of 15 and 18%. With 22% protein they grew at a reduced rate as compared to those on 28%. Protein level of the reproductive diet was shown to be important; low production of eggs and young resulted from levels below 25%. Calcium was found to be even more critical than protein level for reproduction; birds on a winter diet that furnished 145 mg./kg. per day had poor reproductive success the following spring. About 600 mg./kg. of Ca per day was necessary in the reproduction diet. Birds on an intermediate level of Ca (about 0.5% of diet) showed evidence of cumulative deficiency. It was concluded that pheasants receiving levels of Ca no higher than 0.5% in nature might display 'straggling failure' such as has been observed in several midwestern areas.

  8. The influence of protein level in the diet on cannibalism and quality of plumage of layers.

    PubMed

    Ambrosen, T; Petersen, V E

    1997-04-01

    A factorial experiment, with seven levels of protein and seven strains of layers, was conducted to determine the effect of protein level on plumage condition and mortality due to cannibalism. The experiment was carried out with a total of 3,136 layers. The protein content of the feed varied from 11.1 to 19.3%. The experiment revealed that protein levels had an effect (P < 0.0001) on plumage condition, and that the plumage condition also varies (P < 0.01) with strain of layers. A strain by protein levels interaction occurred (P < 0.01) between strains of Leghorn layers, but not between strains of layers of medium body size. The requirement for energy to maintenance was reduced by 10.8 kcal ME per bird per d each time the plumage condition was improved by one point. Mortality due to cannibalism was influenced by protein level (P < 0.001) and strains of layers (P < 0.001). No significant improvement in plumage condition or reduction in cannibalism was obtained with 15.2% or more protein in the feed. The reason for the unsatisfactory plumage condition and the high mortality rate due to cannibalism for the diets low in protein could be inadequate lysine, methionine, and threonine in the diets. But the possibility of an amino acid imbalance in that protein, which is available for the birds after the egg production has taken place, may not be ignored.

  9. Differential protein levels and post-translational modifications in spinal cord injury of the rat.

    PubMed

    Afjehi-Sadat, Leila; Brejnikow, Mika; Kang, Sung Ung; Vishwanath, Vinay; Walder, Nadja; Herkner, Kurt; Redl, Heinz; Lubec, Gert

    2010-03-05

    Although changes in protein expression in spinal cord injury (SCI) would be of pivotal interest, information so far is limited. It was therefore the aim of the study to determine protein levels and post-translational modifications in the early phase following SCI in the rat. SCI was induced in Sprague-Dawley rats and sham operated rats served as controls. A gel-based proteomic approach using two-dimensional gel electrophoresis followed by quantification with specific software and subsequent identification of differentially expressed proteins by nano-ESI-LC-MS/MS was applied. Proteins of several pathways and cascades were dysregulated in SCI: 14-3-3 epsilon protein, dynein light chain 1, and tubulin beta-5 chain showed higher levels in SCI, whereas adenylyl cyclase associated protein 1, dihydropyrimidinase-related protein 2, F-actin capping protein subunit beta, glyceraldehyde-3-phosphate dehydrogenase, stress-induced phosphoprotein 1 and transthyretin showed lower levels in the injured tissue. Post-translational modifications indicated free oxygen radical attack on proteins in SCI. The occurrence of stress is indicated by deranged stress-induced phosphoprotein 1 and signaling abnormalities are reflected by adenylyl cyclase-associated protein 1 and 14-3-3 epsilon protein. The findings propose the involvement of the corresponding cascades and challenge further work into aberrant signaling and oxidative stress in SCI, which may form the basis for experimental intervention for spinal cord trauma.

  10. Importin-β facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels

    PubMed Central

    Schraivogel, Daniel; Schindler, Susann G.; Danner, Johannes; Kremmer, Elisabeth; Pfaff, Janina; Hannus, Stefan; Depping, Reinhard; Meister, Gunter

    2015-01-01

    MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-β pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago – TNRC6 levels. PMID:26170235

  11. A Bayesian framework for cell-level protein network analysis for multivariate proteomics image data

    NASA Astrophysics Data System (ADS)

    Kovacheva, Violet N.; Sirinukunwattana, Korsuk; Rajpoot, Nasir M.

    2014-03-01

    The recent development of multivariate imaging techniques, such as the Toponome Imaging System (TIS), has facilitated the analysis of multiple co-localisation of proteins. This could hold the key to understanding complex phenomena such as protein-protein interaction in cancer. In this paper, we propose a Bayesian framework for cell level network analysis allowing the identification of several protein pairs having significantly higher co-expression levels in cancerous tissue samples when compared to normal colon tissue. It involves segmenting the DAPI-labeled image into cells and determining the cell phenotypes according to their protein-protein dependence profile. The cells are phenotyped using Gaussian Bayesian hierarchical clustering (GBHC) after feature selection is performed. The phenotypes are then analysed using Difference in Sums of Weighted cO-dependence Profiles (DiSWOP), which detects differences in the co-expression patterns of protein pairs. We demonstrate that the pairs highlighted by the proposed framework have high concordance with recent results using a different phenotyping method. This demonstrates that the results are independent of the clustering method used. In addition, the highlighted protein pairs are further analysed via protein interaction pathway databases and by considering the localization of high protein-protein dependence within individual samples. This suggests that the proposed approach could identify potentially functional protein complexes active in cancer progression and cell differentiation.

  12. Effects of dietary protein level on growth and utilization of protein and energy by juvenile mangrove red snapper (Lutjanus argentimaculatus)

    NASA Astrophysics Data System (ADS)

    Ghulam, Abbas; Khalid, Jamil; Rukhsana, Akhtar; Lin, Hong

    2005-01-01

    A feeding trial was conducted in a recirculating water system to investigate the effects of dietary protein levels on growth, feed utilization, hepatosomatic index and liver lipid deposition of juvenile red snapper, Lutjanus argentimaculatus (average initial wet weight 8.0 ± 0.39 g and total length 3.14 ± 0.3 cm). In the experiment, six fishmeal-based diets were formulated to contain various protein levels (20% to 45% in 5% increments), with dietary energy ranging from 2210.7kJ lOOg to 2250.2kJlOOg dry matter. The protein to energy ratios of diets ranged from 8.58 mg protein kJ-1 to 20.03 mg protein kJ-1. Diets were fed for 90d to triplicate groups of fish stocked in 0.128m3 seawater tanks, 25 individuals each. The daily ration of 2% wet body weight was offered to the fish thrice a day. The fish at the end of the study had more than ten-fold (77.0g) increase in weight compared to the initial (8.0g). Fish fed diets of 40% and 45% protein produced significantly (P<0.05) higher weight gain of 77.2g and 76.5g, and specific growth rate (SGR) of 2.65% and 2.62% than those of 67.0 g and 68.3g, and 2.49% and 2.51% of the other diets. The broken-line regression of SGR against dietary protein level yielded an optimum dietary protein requirement of 42.6% (Y=-1.6295 + 0.1114 X 2,P<0.05). Survival remained 100% among groups. Feed conversion ratio decreased from 0.45 for fish fed 20% dietary protein to 0.35 for fish fed 45% dietary protein. Nitrogen intake increased with an increase in dietary protein, which in turn resulted in an increase in nitrogen gain of fish whole body. Fish fed 40% and 45% protein diets showed higher (P<0.05) nitrogen gain (0.27g and 0.26g) than those (0.23g and 025g) fed all other diets. Gross energy intake (GEI) in fish fed 45% protein was lower (600.67kJ) than that (607.97 kJ) of 40% protein diet, though the differences were not statistically significant (P>0.05); GEI ranging from 677.31 kJ to 663.20 kJ at remaining four diets (20% to 35% protein

  13. Maternal folic acid supplementation to dams on marginal protein level alters brain fatty acid levels of their adult offspring.

    PubMed

    Rao, Shobha; Joshi, Sadhana; Kale, Anvita; Hegde, Mahabaleshwar; Mahadik, Sahebarao

    2006-05-01

    Studies on fetal programming of adult diseases have highlighted the importance of maternal nutrition during pregnancy. Folic acid and long-chain essential polyunsaturated fatty acids (LC-PUFAs) have independent effects on fetal growth. However, folic acid effects may also involve alteration of LC-PUFA metabolism. Because marginal deficiency of LC-PUFAs during critical periods of brain growth and development is associated with risks for adult diseases, it is highly relevant to investigate how maternal supplementation of such nutrients can alter brain fatty acid levels. We examined the impact of folic acid supplementation, conventionally used in maternal intervention, on brain essential fatty acid levels and plasma corticosterone concentrations in adult offspring at 11 months of age. Pregnant female rats from 4 groups (6 in each) were fed with casein diets either with 18 g protein/100 g diet (control diet) or treatment diets that were marginal in protein (MP), such as 12 g protein/100 g diet supplemented with 8 mg folic acid (FAS/MP), 12 g protein/100 g diet without folic acid (FAD/MP), or 12 g protein/100 g diet (MP) with 2 mg folic acid. Pups were weaned to a standard laboratory diet with 18 g protein/100 g diet. All male adult offspring in the FAS/MP group showed lower docosahexaenoic acid (P<.05) as compared with control adult offspring (6.04+/-2.28 vs 10.33+/-0.86 g/100 g fatty acids) and higher n-6/n-3 ratio (P<.05). Docosahexaenoic acid levels in FAS/MP adult offspring were also lower (P<.05) when compared with the MP group. Plasma corticosterone concentrations were higher (P<.05) in male adult offspring from the FAS/MP group compared with control as well as the MP adult offspring. Results suggest that maternal folic acid supplementation at MP intake decreased brain docosahexaenoic acid levels probably involving corticosterone increase.

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

    PubMed

    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.

  15. Ubiquitin Ligase Substrate Identification through Quantitative Proteomics at Both the Protein and Peptide Levels

    PubMed Central

    Lee, Kimberly A.; Hammerle, Lisa P.; Andrews, Paul S.; Stokes, Matthew P.; Mustelin, Tomas; Silva, Jeffrey C.; Black, Roy A.; Doedens, John R.

    2011-01-01

    Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His6-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology. PMID:21987572

  16. Efficient monitoring of protein ubiquitylation levels using TUBEs-based microarrays.

    PubMed

    Serna, Sonia; Xolalpa, Wendy; Lang, Valérie; Aillet, Fabienne; England, Patrick; Reichardt, Niels; Rodriguez, Manuel S

    2016-08-01

    Analyzing protein ubiquitylation changes during physiological or pathological processes is challenging due to its high reversibility and dynamic turnover of modified targets. We have developed a protein microarray to assess endogenous ubiquitylation levels from cell cultures, employing tandem ubiquitin-binding entities (TUBEs) with three or four ubiquitin-associated (UBA) domains as capture probes. Adriamycin (ADR)-stimulated MCF7 cells were used to differentiate protein ubiquitylation levels between cells that are sensitive or resistant to ADR treatment. We show that TUBEs-based microarrays can be used for the analysis of cellular processes regulated by ubiquitylation and for the detection of pathologies with aberrant ubiquitylation levels.

  17. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

  18. The impact of carbohydrate and protein level and sources on swine manure foaming properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study explored the impact of swine diet on the composition, methane production potential, and foaming properties of manure. Samples of swine manure were collected from controlled feeding trials with diets varying in protein and carbohydrate levels and sources. Protein sources consisted of corn ...

  19. Optogenetic Stimulation Increases Level of Antiapoptotic Protein Bcl-xL in Neurons.

    PubMed

    Lanshakov, D A; Drozd, U S; Dygalo, N N

    2017-03-01

    The antiapoptotic protein Bcl-xL is associated with several neuroplastic processes such as formation of synapses, regulation of spontaneous and evoked synaptic responses, and release of neurotransmitters. Dependence of expression on activity of neurons is characteristic for many proteins participating in regulation of neuroplasticity. Whether such property is exhibited by the Bcl-xL protein was analyzed using in vivo optogenetic stimulation of hippocampal glutamatergic neurons expressing channelrhodopsin ChR2H134 under CAMKIIa promoter in the adeno-associated viral vector, followed by immunohistochemical determination of the level of Bcl-xL protein in these neurons and surrounding cells. Increase in the level of early response c-Fos protein following illumination with blue light was indicative of activation of these hippocampal neurons. The optogenetic activation of hippocampus resulted in a significant increase in the level of antiapoptotic protein Bcl-xL in the photosensitive neurons as well as in the surrounding cells. The dependence of the level of expression of Bcl-xL protein on the activity of neurons indicates that this protein possesses one more important property that is essential for participation in neuroplastic processes in the brain.

  20. Stable, high-level expression of a type I antifreeze protein in Escherichia coli.

    PubMed

    Solomon, R G; Appels, R

    1999-06-01

    The type I antifreeze proteins are simple amphipathic helical proteins found in abundance in polar fish species, where they act to prevent freezing of internal fluids by a mechanism of noncolligative freezing point depression. Large-scale production of these proteins for research and biotechnological purposes has been hampered by their apparent instability when expressed in heterologous host systems. This has necessitated their production as fusion proteins, in polymeric form, or as proproteins for secretion, with the concomitant necessity for postpurification processing to generate the mature form of the protein. We have successfully expressed a recombinant variant of type I antifreeze protein (rAFP) in Escherichia coli using the inducible T7 polymerase transcription expression system. The rAFP contains five copies of the 11 amino acid ice-binding repeat motif found in all type I antifreeze proteins. The protein accumulates to high levels intracellularly in the form of inclusion bodies, with no apparent degradation by the cellular proteolytic machinery. We have devised a simple and rapid purification protocol for this recombinant type I antifreeze protein which does not require cellular fractionation, purification of the inclusion bodies, or chromatographic steps. This protocol may be of general use for this class of protein. The protein displays all three activities common to these proteins: recrystallization inhibition, noncolligative freezing point depression, and modification of the morphology of single ice crystals in solution.

  1. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

  2. Altered protein levels in the isolated extracellular matrix of failing human hearts with dilated cardiomyopathy.

    PubMed

    DeAguero, Joshua L; McKown, Elizabeth N; Zhang, Liwen; Keirsey, Jeremy; Fischer, Edgar G; Samedi, Von G; Canan, Benjamin D; Kilic, Ahmet; Janssen, Paul M L; Delfín, Dawn A

    Dilated cardiomyopathy (DCM) is associated with extensive pathological cardiac remodeling and involves numerous changes in the protein expression profile of the extracellular matrix of the heart. We obtained seven human, end-stage, failing hearts with DCM (DCM-failing) and nine human, nonfailing donor hearts and compared their extracellular matrix protein profiles. We first showed that the DCM-failing hearts had indeed undergone extensive remodeling of the left ventricle myocardium relative to nonfailing hearts. We then isolated the extracellular matrix from a subset of these hearts and performed a proteomic analysis on the isolated matrices. We found that the levels of 26 structural proteins were altered in the DCM-failing isolated cardiac extracellular matrix compared to nonfailing isolated cardiac extracellular matrix. Overall, most of the extracellular matrix proteins showed reduced levels in the DCM-failing hearts, while all of the contractile proteins showed increased levels. There was a mixture of increased and decreased levels of cytoskeletal and nuclear transport proteins. Using immunoprobing, we verified that collagen IV (α2 and α6 isoforms), zyxin, and myomesin protein levels were reduced in the DCM-failing hearts. We expect that these data will add to the understanding of the pathology associated with heart failure with DCM.

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

    PubMed Central

    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

  4. Fragile X mental retardation protein regulates the levels of scaffold proteins and glutamate receptors in postsynaptic densities.

    PubMed

    Schütt, Janin; Falley, Katrin; Richter, Dietmar; Kreienkamp, Hans-Jürgen; Kindler, Stefan

    2009-09-18

    Functional absence of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a hereditary form of mental retardation characterized by a change in dendritic spine morphology. The RNA-binding protein FMRP has been implicated in regulating postsynaptic protein synthesis. Here we have analyzed whether the abundance of scaffold proteins and neurotransmitter receptor subunits in postsynaptic densities (PSDs) is altered in the neocortex and hippocampus of FMRP-deficient mice. Whereas the levels of several PSD components are unchanged, concentrations of Shank1 and SAPAP scaffold proteins and various glutamate receptor subunits are altered in both adult and juvenile knock-out mice. With the exception of slightly increased hippocampal SAPAP2 mRNA levels in adult animals, altered postsynaptic protein concentrations do not correlate with similar changes in total and synaptic levels of corresponding mRNAs. Thus, loss of FMRP in neurons appears to mainly affect the translation and not the abundance of particular brain transcripts. Semi-quantitative analysis of RNA levels in FMRP immunoprecipitates showed that in the mouse brain mRNAs encoding PSD components, such as Shank1, SAPAP1-3, PSD-95, and the glutamate receptor subunits NR1 and NR2B, are associated with FMRP. Luciferase reporter assays performed in primary cortical neurons from knock-out and wild-type mice indicate that FMRP silences translation of Shank1 mRNAs via their 3'-untranslated region. Activation of metabotropic glutamate receptors relieves translational suppression. As Shank1 controls dendritic spine morphology, our data suggest that dysregulation of Shank1 synthesis may significantly contribute to the abnormal spine development and function observed in brains of fragile X syndrome patients.

  5. Comparison of total protein and phospholipase A(2) levels in individual coralsnake venoms.

    PubMed

    Kopper, Randall A; Harper, George R; Zimmerman, Sloane; Hook, Jessica

    2013-12-15

    Studies of differences or changes in venom protein levels or enzymatic activities have significance only if contrasted to the normal variations between individual snakes. This study involves the analysis and comparison of venom from 13 individual Texas coralsnakes (Micrurus tener tener) in order to detect differences in the volume, total protein concentration, electrophoretic profile, and PLA2 enzyme activity. A significant inverse correlation between venom volume and total protein concentration was found. Although the 13 venoms were indistinguishable from their electrophoretic protein profiles, phospholipase A2 enzymatic activities varied considerably.

  6. How to talk about protein-level false discovery rates in shotgun proteomics.

    PubMed

    The, Matthew; Tasnim, Ayesha; Käll, Lukas

    2016-09-01

    A frequently sought output from a shotgun proteomics experiment is a list of proteins that we believe to have been present in the analyzed sample before proteolytic digestion. The standard technique to control for errors in such lists is to enforce a preset threshold for the false discovery rate (FDR). Many consider protein-level FDRs a difficult and vague concept, as the measurement entities, spectra, are manifestations of peptides and not proteins. Here, we argue that this confusion is unnecessary and provide a framework on how to think about protein-level FDRs, starting from its basic principle: the null hypothesis. Specifically, we point out that two competing null hypotheses are used concurrently in today's protein inference methods, which has gone unnoticed by many. Using simulations of a shotgun proteomics experiment, we show how confusing one null hypothesis for the other can lead to serious discrepancies in the FDR. Furthermore, we demonstrate how the same simulations can be used to verify FDR estimates of protein inference methods. In particular, we show that, for a simple protein inference method, decoy models can be used to accurately estimate protein-level FDRs for both competing null hypotheses.

  7. Possible role for increased C4b-binding-protein level in acquired protein S deficiency in type I diabetes.

    PubMed

    Ceriello, A; Giugliano, D; Quatraro, A; Marchi, E; Barbanti, M; Lefebvre, P

    1990-04-01

    In this study, total protein S (PS) immunological levels, free-PS and C4b-binding-protein (C4bBP) concentrations, and PS functional activity were investigated in insulin-dependent (type I) diabetic patients and compared with nondiabetic subjects. Mean total PS antigen concentration was not different between diabetic patients and nondiabetic subjects, whereas free-PS levels and PS functional activity were significantly reduced in diabetic patients. C4bBP was increased in diabetic patients and correlated with HbA1 levels. This study shows that type I diabetic patients have depressed free PS and PS activity despite the presence of normal total PS concentration and suggests that this phenomenon is probably linked to the increase of circulating C4bBP.

  8. Effect of energy and protein levels on nutrient utilization and their requirements in growing Murrah buffaloes.

    PubMed

    Prusty, Sonali; Kundu, Shivlal Singh; Mondal, Goutam; Sontakke, Umesh; Sharma, Vijay Kumar

    2016-04-01

    To evaluate different levels of energy and protein for optimum growth of Murrah male buffalo calves, a growth trial (150 days) was conducted on 30 calves (body weight 202.5 ± 6.8 kg). Six diets were formulated to provide 90, 100 and 110% protein level and 90 and 110% energy level requirements for buffalo calves, derived from ICAR 2013 recommendations for buffaloes. The crude protein (CP) intake was increased with higher dietary CP, whereas no effect of energy levels or interaction between protein and energy was observed on CP intake. There were significant effects (P < 0.01) of the interaction between protein and energy (P < 0.05) on metabolizable energy (ME) intake. The digestibility of dry matter (DM), organic matter (OM) and non-fibrous carbohydrate (NFC) was higher (P < 0.0001) in high-energy groups compared to low-energy groups. The CP digestibility increased with the increased CP and ME of the rations. The absorbed N was improved linearly with an increased level of dietary CP, whereas the N retention was similar among all the groups distributed as per different energy or protein levels. The nutrient intake (protein or energy) per kg body weight (BW)(0.75) at various fortnight intervals was regressed linearly from the average daily gain (ADG) per kg BW(0.75). By setting the average daily gain at zero in the developed regression equation, a maintenance requirement was obtained, i.e. 133.1 kcal ME, 6.45 g CP and 3.95 g metabolizable protein (MP) per kg BW(0.75). Requirement for growth was 6.12 kcal ME, 0.46 g CP and 0.32 g MP per kg BW(0.75) per day. Metabolizable amino acid requirement was estimated from partitioning of MP intake and ADG. The ME requirements were lower, whereas the MP requirement of Murrah buffaloes was higher than ICAR (2013) recommendations.

  9. A Ketogenic Diet Increases Brown Adipose Tissue Mitochondrial Proteins and UCP1 Levels in Mice

    PubMed Central

    Srivastava, Shireesh; Baxa, Ulrich; Niu, Gang; Chen, Xiaoyuan; Veech, Richard L.

    2013-01-01

    We evaluated the effects of feeding a ketogenic diet (KD) for a month on general physiology with emphasis on brown adipose tissue (BAT) in mice. KD did not reduce the caloric intake, or weight or lipid content of BAT. Relative epididymal fat pads were 40% greater in the mice fed the KD (P = 0.06) while leptin was lower (P < 0.05). Blood glucose levels were 30% lower while D-β-hydroxybutyrate levels were about 3.5-fold higher in the KD group. Plasma insulin and leptin levels in the KD group were about half of that of the mice fed NIH-31 pellets (chow group). Median mitochondrial size in the inter-scapular BAT (IBAT) of the KD group was about 60% greater, whereas the median lipid droplet size was about half of that in the chow group. Mitochondrial oxidative phosphorylation proteins were increased (1.5–3-fold) and the uncoupling protein 1 levels were increased by threefold in mice fed the KD. The levels of PPARγ, PGC-1α, and Sirt1 in KD group were 1.5–3-fold while level of Sirt3 was about half of that in the chow-fed group. IBAT cyclic AMP levels were 60% higher in the KD group and cAMP response element binding protein was 2.5-fold higher, suggesting increased sympathetic system activity. These results demonstrate that a KD can also increase BAT mitochondrial size and protein levels. PMID:23233333

  10. Burial Level Change Defines a High Energetic Relevance for Protein Binding Interfaces.

    PubMed

    Li, Zhenhua; He, Ying; Wong, Limsoon; Li, Jinyan

    2015-01-01

    Protein-protein interfaces defined through atomic contact or solvent accessibility change are widely adopted in structural biology studies. But, these definitions cannot precisely capture energetically important regions at protein interfaces. The burial depth of an atom in a protein is related to the atom's energy. This work investigates how closely the change in burial level of an atom/residue upon complexation is related to the binding. Burial level change is different from burial level itself. An atom deeply buried in a monomer with a high burial level may not change its burial level after an interaction and it may have little burial level change. We hypothesize that an interface is a region of residues all undergoing burial level changes after interaction. By this definition, an interface can be decomposed into an onion-like structure according to the burial level change extent. We found that our defined interfaces cover energetically important residues more precisely, and that the binding free energy of an interface is distributed progressively from the outermost layer to the core. These observations are used to predict binding hot spots. Our approach's F-measure performance on a benchmark dataset of alanine mutagenesis residues is much superior or similar to those by complicated energy modeling or machine learning approaches.

  11. Proteomic analysis of day-night variations in protein levels in the rat pineal gland.

    PubMed

    Møller, Morten; Sparre, Thomas; Bache, Nicolai; Roepstorff, Peter; Vorum, Henrik

    2007-06-01

    The pineal gland secretes the hormone melatonin. This secretion exhibits a circadian rhythm with a zenith during night and a nadir during day. We have performed proteome analysis of the superficial pineal gland in rats during daytime and nighttime. The proteins were extracted and subjected to 2-DE. Of 1747 protein spots revealed by electrophoresis, densitometric analysis showed the up-regulation of 25 proteins during nighttime and of 35 proteins during daytime. Thirty-seven of the proteins were identified by MALDI-TOF MS. The proteins up-regulated during the night are involved in the Krebs cycle, energy transduction, calcium binding, and intracellular transport. During the daytime, enzymes involved in glycolysis, electron transport, and also the Krebs cycle were up-regulated as well as proteins taking part in RNA binding and RNA processing. Our data show a prominent day-night variation of the protein levels in the rat pineal gland. Some proteins are up-regulated during the night concomitant with the melatonin secretion of the gland. Other proteins are up-regulated during the day indicating a pineal metabolism not related to the melatonin synthesis.

  12. Exceptionally high heterologous protein levels in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences.

    PubMed

    De Jaeger, Geert; Angenon, Geert; Depicker, Ann

    2003-01-01

    Seeds are concentrated sources of protein and thus may be ideal 'bioreactors' for the production of heterologous proteins. For this application, strong seed-specific expression signals are required. A set of expression cassettes were designed using 5' and 3' regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) from Phaseolus vulgaris, and evaluated for the production of heterologous proteins in dicotyledonous plant species. A murine single-chain variable fragment (scFv) was chosen as model protein because of the current industrial interest to produce antibodies and derived fragments in crops. Because the highest scFv accumulation in seed had previously been achieved in the endoplasmic reticulum (ER), the scFv-encoding sequence was provided with signal sequences for accumulation in the ER. Transgenic Arabidopsis seed stocks, expressing the scFv under control of the 35S promoter, contained scFv accumulation levels in the range of 1% of total soluble protein (TSP). However, the seed storage promoter constructs boosted the scFv to exceptionally high levels. Maximum scFv levels were obtained in homozygous seed stocks, being 12.5% of TSP under control of the arc5-I regulatory sequences and even up to 36.5% of TSP upon replacing the arc5-I promoter by the beta-phaseolin promoter of Phaseolus vulgaris. Even at such very high levels, the scFv proteins retain their full antigen-binding activity. Moreover, the presence of very high scFv levels has only minory effects on seed germination and no effect on seed production. These results demonstrate that the expression levels of arcelin 5-I and beta-phaseolin seed storage protein genes can be transferred to heterologous proteins, giving exceptionally high levels of heterologous proteins, which can be of great value for the molecular farming industry by raising production yield and lowering bio-mass production and purification costs. Finally, the feasibility of heterologous protein production using the

  13. Enteral glutamine stimulates protein synthesis and decreases ubiquitin mRNA level in human gut mucosa.

    PubMed

    Coëffier, Moïse; Claeyssens, Sophie; Hecketsweiler, Bernadette; Lavoinne, Alain; Ducrotté, Philippe; Déchelotte, Pierre

    2003-08-01

    Effects of glutamine on whole body and intestinal protein synthesis and on intestinal proteolysis were assessed in humans. Two groups of healthy volunteers received in a random order enteral glutamine (0.8 mmol.kg body wt(-1)x h(-1)) compared either to saline or isonitrogenous amino acids. Intravenous [2H5]phenylalanine and [13C]leucine were simultaneously infused. After gas chromatography-mass spectrometry analysis, whole body protein turnover was estimated from traced plasma amino acid fluxes and the fractional synthesis rate (FSR) of gut mucosal protein was calculated from protein and intracellular phenylalanine and leucine enrichments in duodenal biopsies. mRNA levels for ubiquitin, cathepsin D, and m-calpain were analyzed in biopsies by RT-PCR. Glutamine significantly increased mucosal protein FSR compared with saline. Glutamine and amino acids had similar effects on FSR. The mRNA level for ubiquitin was significantly decreased after glutamine infusion compared with saline and amino acids, whereas cathepsin D and m-calpain mRNA levels were not affected. Enteral glutamine stimulates mucosal protein synthesis and may attenuate ubiquitin-dependent proteolysis and thus improve protein balance in human gut.

  14. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes.

    PubMed

    Soltani, Mohammad; Vargas-Garcia, Cesar A; Antunes, Duarte; Singh, Abhyudai

    2016-08-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells.

  15. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes

    PubMed Central

    Soltani, Mohammad; Vargas-Garcia, Cesar A.; Antunes, Duarte; Singh, Abhyudai

    2016-01-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells. PMID:27536771

  16. Controlled levels of protein modification through a chromatography-mediated bioconjugation

    SciTech Connect

    Kwant, Richard L.; Jaffe, Jake; Palmere, Peter J.; Francis, Matthew B.

    2015-02-27

    Synthetically modified proteins are increasingly finding applications as well-defined scaffolds for materials. In practice it remains difficult to construct bioconjugates with precise levels of modification because of the limited number of repeated functional groups on proteins. This article describes a method to control the level of protein modification in cases where there exist multiple potential modification sites. A protein is first tagged with a handle using any of a variety of modification chemistries. This handle is used to isolate proteins with a particular number of modifications via affinity chromatography, and then the handle is elaborated with a desired moiety using an oxidative coupling reaction. This method results in a sample of protein with a well-defined number of modifications, and we find it particularly applicable to systems like protein homomultimers in which there is no way to discern between chemically identical subunits. We demonstrate the use of this method in the construction of a protein-templated light-harvesting mimic, a type of system which has historically been difficult to make in a well-defined manner.

  17. Controlled levels of protein modification through a chromatography-mediated bioconjugation

    DOE PAGES

    Kwant, Richard L.; Jaffe, Jake; Palmere, Peter J.; ...

    2015-02-27

    Synthetically modified proteins are increasingly finding applications as well-defined scaffolds for materials. In practice it remains difficult to construct bioconjugates with precise levels of modification because of the limited number of repeated functional groups on proteins. This article describes a method to control the level of protein modification in cases where there exist multiple potential modification sites. A protein is first tagged with a handle using any of a variety of modification chemistries. This handle is used to isolate proteins with a particular number of modifications via affinity chromatography, and then the handle is elaborated with a desired moiety usingmore » an oxidative coupling reaction. This method results in a sample of protein with a well-defined number of modifications, and we find it particularly applicable to systems like protein homomultimers in which there is no way to discern between chemically identical subunits. We demonstrate the use of this method in the construction of a protein-templated light-harvesting mimic, a type of system which has historically been difficult to make in a well-defined manner.« less

  18. High levels of protein expression using different mammalian CMV promoters in several cell lines.

    PubMed

    Xia, Wei; Bringmann, Peter; McClary, John; Jones, Patrick P; Manzana, Warren; Zhu, Ying; Wang, Soujuan; Liu, Yi; Harvey, Susan; Madlansacay, Mary Rose; McLean, Kirk; Rosser, Mary P; MacRobbie, Jean; Olsen, Catherine L; Cobb, Ronald R

    2006-01-01

    With the recent completion of the human genome sequencing project, scientists are faced with the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. Equally as important is to produce milligram quantities of the therapeutically relevant gene products as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications including proper protein folding and glycosylation. In response to the needs described above, we investigated the protein expression levels driven by the human CMV in the presence or absence of intron A, the mouse and rat CMV promoters with intron A, and the MPSV promoter in plasmid expression vectors. We evaluated the different promoters using an in-house plasmid vector backbone. The protein expression levels of four genes of interest driven by these promoters were evaluated in HEK293EBNA and CHO-K1 cells. Stable and transient transfected cells were utilized. In general, the full-length human CMV, in the presence of intron A, gave the highest levels of protein expression in transient transfections in both cell lines. However, the MPSV promoter resulted in the highest levels of stable protein expression in CHO-K1 cells. Using the CMV driven constitutive promoters in the presence of intron A, we have been able to generate >10 microg/ml of recombinant protein using transient transfections.

  19. Multi-level machine learning prediction of protein–protein interactions in Saccharomyces cerevisiae

    PubMed Central

    Zubek, Julian; Tatjewski, Marcin; Boniecki, Adam; Mnich, Maciej; Basu, Subhadip

    2015-01-01

    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). PMID:26157620

  20. A two level hierarchical model of protein retention in ion exchange chromatography.

    PubMed

    Salvalaglio, Matteo; Paloni, Matteo; Guelat, Bertrand; Morbidelli, Massimo; Cavallotti, Carlo

    2015-09-11

    Predicting protein retention in ion exchange chromatography (IEX) from first principles is a fascinating perspective. In this work a two level hierarchical modeling strategy is proposed in order to calculate protein retention factors. Model predictions are tested against experimental data measured for Lysozyme and Chymotrypsinogen A in IEX columns as a function of ionic strength and pH. At the highest level of accuracy Molecular Dynamics (MD) simulations in explicit water are used to determine the interaction free energy between each of the two proteins and the IEX stationary phase for a reference pH and ionic strength. At a lower level of accuracy a linear response model based on an implicit treatment of solvation and adopting a static protein structure is used to calculate interaction free energies for the full range of pHs and ionic strengths considered. A scaling coefficient, determined comparing MD and implicit solvent simulations, is then introduced in order to correct the linear response model for errors induced by the adoption of a static protein structure. The calculated free energies are then used to compute protein retention factors, which can be directly compared with experimental data. The possibility to introduce a third level of accuracy is explored testing the predictions of a semiempirical model. A quantitative agreement between the predicted and measured protein retention factors is obtained using the coupled MD-linear response models, supporting the reliability of the proposed approach. The model allows quantifying the electrostatic, van der Waals, and conformational contributions to the interaction free energies. A good agreement between experiments and model is obtained also using the semiempirical model that, although requiring parameterization over higher level models or experimental data, proves to be useful in order to rapidly determine protein retention factors across wide pH and ionic strength ranges as it is computationally inexpensive.

  1. Activity and circadian rhythm influence synaptic Shank3 protein levels in mice.

    PubMed

    Sarowar, Tasnuva; Chhabra, Resham; Vilella, Antonietta; Boeckers, Tobias M; Zoli, Michele; Grabrucker, Andreas M

    2016-09-01

    Various recent studies revealed that the proteins of the Shank family act as major scaffold organizing elements in the post-synaptic density of excitatory synapses and that their expression level is able to influence synapse formation, maturation and ultimately brain plasticity. An imbalance in Shank3 protein levels has been associated with a variety of neuropsychological and neurodegenerative disorders including autism spectrum disorders and Phelan-McDermid syndrome. Given that sleep disorders and low melatonin levels are frequently observed in autism spectrum disorders, and that circadian rhythms may be able to modulate Shank3 signaling and thereby synaptic function, here, we performed in vivo studies on CBA mice using protein biochemistry to investigate the synaptic expression levels of Shank3α during the day in different brain regions. Our results show that synaptic Shank3 protein concentrations exhibit minor oscillations during the day in hippocampal and striatal brain regions that correlate with changes in serum melatonin levels. Furthermore, as circadian rhythms are tightly connected to activity levels in mice, we increased physical activity using running wheels. The expression of Shank3α increases rapidly by induced activity in thalamus and cortex, but decreases in striatum, superimposing the circadian rhythms of different brain regions. We conclude that synaptic Shank3 proteins build highly dynamic platforms that are modulated by the light:dark cycles but even more so driven by activity. Using wild-type CBA mice, we show that Shank3 is a highly dynamic and activity-regulated protein at synapses. In the hippocampus, changes in synaptic Shank3 levels are influenced by circadian rhythm/melatonin concentration, while running activity increases and decreases levels of Shank3 in the cortex and striatum respectively.

  2. Genetic evidence that oxidative derivatives of retinoic acid are not involved in retinoid signaling during mouse development.

    PubMed

    Niederreither, Karen; Abu-Abed, Suzan; Schuhbaur, Brigitte; Petkovich, Martin; Chambon, Pierre; Dollé, Pascal

    2002-05-01

    Retinoic acid, the active derivative of vitamin A (retinol), is a hormonal signaling molecule that acts in developing and adult tissues. The Cyp26a1 (cytochrome p450, 26) protein metabolizes retinoic acid into more polar hydroxylated and oxidized derivatives. Whether some of these derivatives are biologically active metabolites has been debated. Cyp26a1(-/-) mouse fetuses have lethal morphogenetic phenotypes mimicking those generated by excess retinoic acid administration, indicating that human CYP26A1 may be essential in controlling retinoic acid levels during development. This hypothesis suggests that the Cyp26a1(-/-) phenotype could be rescued under conditions in which embryonic retinoic acid levels are decreased. We show that Cyp26a1(-/-) mice are phenotypically rescued by heterozygous disruption of Aldh1a2 (also known as Raldh2), which encodes a retinaldehyde dehydrogenase responsible for the synthesis of retinoic acid during early embryonic development. Aldh1a2 haploinsufficiency prevents the appearance of spina bifida and rescues the development of posterior structures (sacral/caudal vertebrae, hindgut, urogenital tract), while partly preventing cervical vertebral transformations and hindbrain pattern alterations in Cyp26a1(-/-) mice. Thus, some of these double-mutant mice can reach adulthood. This study is the first report of a mutation acting as a dominant suppressor of a lethal morphogenetic mutation in mammals. We provide genetic evidence that ALDH1A2 and CYP26A1 activities concurrently establish local embryonic retinoic acid levels that must be finely tuned to allow posterior organ development and to prevent spina bifida.

  3. Reduced Activity-Dependent Protein Levels in a Mouse Model of the Fragile X Premutation

    PubMed Central

    von Leden, Ramona E.; Curley, Lindsey C.; Greenberg, Gian D.; Hunsaker, Michael R.; Willemsen, Rob; Berman, Robert F.

    2014-01-01

    Environmental enrichment results in increased levels of Fmrp in brain and increased dendritic complexity. The present experiment evaluated activity-dependent increases in Fmrp levels in the motor cortex in response to training on a skilled forelimb reaching task in the CGG KI mouse model of the fragile X premutation. Fmrp, Arc, and c-Fos protein levels were quantified by Western blot in the contralateral motor cortex of mice following training to reach for sucrose pellets with a non-preferred paw and compared to levels in the ipsilateral motor cortex. After training, all mice showed increases in Fmrp, Arc, and c-Fos protein levels in the contralateral compared to the ipsilateral hemisphere; however, the increase in CGG KI mice was less than wildtype mice. Increases in Fmrp and Arc proteins scaled with learning, whereas this relationship was not observed with the c-Fos levels. These data suggest the possibility that reduced levels of activity-dependent proteins associated with synaptic plasticity such as Fmrp and Arc may contribute to the neurocognitive phenotype reported in the CGG KI mice and the fragile X premutation. PMID:24462720

  4. Reduced activity-dependent protein levels in a mouse model of the fragile X premutation.

    PubMed

    von Leden, Ramona E; Curley, Lindsey C; Greenberg, Gian D; Hunsaker, Michael R; Willemsen, Rob; Berman, Robert F

    2014-03-01

    Environmental enrichment results in increased levels of Fmrp in brain and increased dendritic complexity. The present experiment evaluated activity-dependent increases in Fmrp levels in the motor cortex in response to training on a skilled forelimb reaching task in the CGG KI mouse model of the fragile X premutation. Fmrp, Arc, and c-Fos protein levels were quantified by Western blot in the contralateral motor cortex of mice following training to reach for sucrose pellets with a non-preferred paw and compared to levels in the ipsilateral motor cortex. After training, all mice showed increases in Fmrp, Arc, and c-Fos protein levels in the contralateral compared to the ipsilateral hemisphere; however, the increase in CGG KI mice was less than wildtype mice. Increases in Fmrp and Arc proteins scaled with learning, whereas this relationship was not observed with the c-Fos levels. These data suggest the possibility that reduced levels of activity-dependent proteins associated with synaptic plasticity such as Fmrp and Arc may contribute to the neurocognitive phenotype reported in the CGG KI mice and the fragile X premutation.

  5. AFM visualization at a single-molecule level of denaturated states of proteins on graphite.

    PubMed

    Barinov, Nikolay A; Prokhorov, Valery V; Dubrovin, Evgeniy V; Klinov, Dmitry V

    2016-10-01

    Different graphitic materials are either already used or believed to be advantageous in biomedical and biotechnological applications, e.g., as biomaterials or substrates for sensors. Most of these applications or associated important issues, such as biocompatibility, address the problem of adsorption of protein molecules and, in particular the conformational state of the adsorbed protein molecule on graphite. High-resolution AFM demonstrates highly oriented pyrolytic graphite (HOPG) induced denaturation of four proteins of blood plasma, such as ferritin, fibrinogen, human serum albumin (HSA) and immunoglobulin G (IgG), at a single molecule level. Protein denaturation is accompanied by the decrease of the heights of protein globules and spreading of the denatured protein fraction on the surface. In contrast, the modification of HOPG with the amphiphilic oligoglycine-hydrocarbon derivative monolayer preserves the native-like conformation and provides even more mild conditions for the protein adsorption than typically used mica. Protein unfolding on HOPG may have universal character for "soft" globular proteins.

  6. Alcohol-binding sites in distinct brain proteins: the quest for atomic level resolution.

    PubMed

    Howard, Rebecca J; Slesinger, Paul A; Davies, Daryl L; Das, Joydip; Trudell, James R; Harris, R Adron

    2011-09-01

    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 neurotransmitter-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.

  7. Glycosylated serum protein level as a screening and diagnostic test for gestational diabetes mellitus.

    PubMed

    Bourgeois, F J; Harbert, G M; Paulsen, E P; Thiagarajah, S

    1986-09-01

    Glycosylated serum protein assay was examined as an alternative to standard glucose screening and glucose tolerance testing. In a comparison of two groups of gravid women having abnormal 1-hour 50 gm glucose screening tests, there was no difference in glycosylated protein level in the group with abnormal glucose tolerance test results (9.4% +/- 2.0%, mean +/- SD; n = 8) versus normal results (9.2% +/- 1.07%, mean +/- SD; n = 11). Furthermore, correlation of glycosylated serum protein level with glucose screening test results was poor (r = 0.185, p = 0.23, n = 17). Glycosylated serum protein assay is not useful in detecting mild metabolic aberrations associated with gestational diabetes.

  8. Heavy metals chromium and neodymium reduced phosphorylation level of heat shock protein 27 in human keratinocytes.

    PubMed

    Zhang, Qihao; Zhang, Lei; Xiao, Xue; Su, Zhijian; Zou, Ping; Hu, Hao; Huang, Yadong; He, Qing-Yu

    2010-06-01

    Heavy metals may exert their acute and chronic effects on the human skin through stress signals. In the present study, 2DE-based proteomics was used to analyze the protein expression in human keratinocytes exposed to heavy metals, chromium and neodymium, and 10 proteins with altered expression were identified. Among these proteins, small heat shock protein 27 (HSP27) was up-regulated significantly and the up-regulation was validated by Western blot and immunofluorescence. In addition, the mRNA expression level of HSP27 markedly increased as detected by quantitative PCR. More interestingly, the ratio of phosphorylated HSP27 and total HSP27 significantly decreased in keratinocytes treated with the heavy metals. These findings suggested that heavy metals reduced the phosphorylation level of HSP27, and that the ratio of p-HSP27 and HSP27 may represent a potential marker or additional endpoint for the hazard assessment of skin irritation caused by chemical products.

  9. High MMP-1, MMP-2, and MMP-9 protein levels in osteoarthritis.

    PubMed

    Zeng, G Q; Chen, A B; Li, W; Song, J H; Gao, C Y

    2015-11-23

    Our study examined the relationship between the expression of matrix metalloproteinases (MMP)-1, MMP-2, and MMP-9 proteins and the pathogenesis of osteoarthritis (OA). We employed rigorous inclusion and exclusion criteria in computer-based bibliographic databases to extract published studies relevant to this investigation. The STATA 12.0 software was used for the statistical analyses. A total of 1408 studies were initially searched, and 10 studies with 458 OA patients and 295 healthy controls were included in this meta-analysis. The meta-analysis results suggested that the protein levels of MMP-1, MMP-2, and MMP-9 were higher in patients with OA than those in the control group. A subgroup analysis according to ethnicity showed that the protein levels of MMP-1 and MMP-2 were higher in Asian patients with OA than in controls. Caucasians showed no statistically significant differences in protein expression of MMP-1 and MMP-2 between the OA patient group and the control group. Interestingly, the protein levels of MMP-9 in patients with OA were higher than those in the control group in both Asians and Caucasians. A sample-source analysis suggested that the serum levels of MMP-2 and MMP-9 proteins were higher in patients with OA than in controls, while MMP-1 and MMP-9 protein expressions were higher in the synovial joint fluid of patients with OA than in controls. In conclusion, our meta-analysis results suggested that the increased expression of MMP-1, MMP-2, and MMP-9 proteins might be associated with the pathogenesis of OA.

  10. The significance of C-reactive protein levels in women with premature rupture of membranes.

    PubMed

    Ismail, M A; Zinaman, M J; Lowensohn, R I; Moawad, A H

    1985-02-15

    In a prospective study of 100 patients with preterm premature rupture of membranes, clinical chorioamnionitis was present in 18 and histologic chorioamnionitis was present in 63. Patients who were managed conservatively for premature rupture of membranes were monitored by C-reactive protein determination, white blood cell and differential counts, maternal temperature, and fetal heart tone. C-reactive protein was measured nephelometrically (Immuno-chemistry Analyzer II, Beckman). Elevated C-reactive protein levels correlated well with both the pathologic and the clinical diagnosis of chorioamnionitis. Elevated C-reactive protein levels (at least 12 to 24 hours before delivery) were more sensitive than other standard laboratory or clinical tests in predicting chorioamnionitis both by clinical and pathologic criteria. When C-reactive protein values were normal, clinical chorioamnionitis was rarely found, whereas pathologically diagnosed chorioamnionitis was found half of the time. We conclude that although the C-reactive protein level is a very sensitive predictor of infectious morbidity in premature rupture of membranes, its specificity is not high.

  11. Expression of fas protein on CD4+T cells irradiated by low level He-Ne

    NASA Astrophysics Data System (ADS)

    Nie, Fan; Zhu, Jing; Zhang, Hui-Guo

    2005-07-01

    Objective: To investigate the influence on the Expression of Fas protein on CD4+ T cells irradiated by low level He-Ne laser in the cases of psoriasis. Methods:the expression of CD4+ T Fas protein was determined in the casee of psoriasis(n=5) pre and post-low level laser irradiation(30 min、60min and 120min)by flow cytometry as compared withthe control(n=5). Results:In the cases of psoriasis,the expression of CD4+T FAS protein 21.4+/-3.1% was increased significantly than that of control group 16.8+/-2.1% pre-irradiation, p<0.05in the control,there is no difference between pre and post- irradiation,p>0.05in the cases , the expression of CD4+T Fas protein wae positively corelated to the irradiation times, when the energy density arrived to 22.92J/cm2(60 minutes)and 45.84J/cm2(120minutes), the expression of CD4+ T Fas protein was increased significantly as compared with pre-irradiation,p<0.05.Conclusion: The expression of CD4+T Fas protein may be increased by low level He-Ne laser irradiation ,the uncontrolled status of apoptosis could be corrected.

  12. Low levels of protein Z are associated with HELLP syndrome and its severity.

    PubMed

    Kaygusuz, Isik; Firatli-Tuglular, Tulin; Toptas, Tayfur; Ugurel, Vedat; Demir, Muzaffer

    2011-04-01

    Protein Z (PZ) was found to be associated with pregnancy complications. There are no data implying an association between hemolysis (H), elevated liver enzymes (EL), and low platelet counts (LP) (HELLP) syndrome and changes in plasma levels of PZ. The aim of this study is to investigate whether HELLP syndrome is associated with plasma concentrations of PZ. Protein Z levels in 29 women with HELLP syndrome were compared with 29 healthy, nulliparous and 25 normal pregnant women. The median PZ levels in patients with HELLP syndrome were found to be significantly lower than those of pregnant women. No significant difference was found between HELLP and healthy groups. Protein Z levels correlated with platelet counts, lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) levels in patients with HELLP syndrome. Median PZ level was higher in partial HELLP than in complete HELLP. We calculated 1330 ng/mL as a cutoff value for PZ level to discriminate HELLP syndrome from normal pregnancy. Low PZ levels are associated with the pathobiology of HELLP syndrome.

  13. Temperature and dietary starch level affected protein but not starch digestibility in gilthead sea bream juveniles.

    PubMed

    Couto, A; Enes, P; Peres, H; Oliva-Teles, A

    2012-06-01

    A study was carried out with gilthead sea bream juveniles to assess the effect of water temperature (18 and 25°C) and dietary pregelatinized starch level (10, 20 and 30%) on digestibility of protein and starch and on the activity of proteolytic and amylolytic enzymes. ADC of pregelatinized starch was very high (>99%) irrespectively of dietary inclusion level, and it was not affected by water temperature. ADC of protein was also high (>90%) but improved at the higher water temperature. Dietary starch interacted with protein digestibility, which decreased as dietary starch level increased. Temperature affected both acid and basic protease activities, with acid protease activity being higher at 25°C and basic protease activity being higher at 18°C. However, total proteolytic activity and amylase activities were not affected by water temperature. Dietary carbohydrate exerted no effect on proteolytic or amylolitic activities. It is concluded that gilthead sea bream juveniles digest pregelatinized starch very efficiently irrespective of water temperature, due to adjustments of amylase activity to cope with temperature differences. Pregelatinized starch interacts negatively with protein digestibility, with the ADC of protein decreasing as dietary starch levels increase.

  14. Differential Acute and Chronic Effects of Leptin on Hypothalamic Astrocyte Morphology and Synaptic Protein Levels

    PubMed Central

    García-Cáceres, Cristina; Fuente-Martín, Esther; Burgos-Ramos, Emma; Granado, Miriam; Frago, Laura M.; Barrios, Vicente; Horvath, Tamas

    2011-01-01

    Astrocytes participate in neuroendocrine functions partially through modulation of synaptic input density in the hypothalamus. Indeed, glial ensheathing of neurons is modified by specific hormones, thus determining the availability of neuronal membrane space for synaptic inputs, with the loss of this plasticity possibly being involved in pathological processes. Leptin modulates synaptic inputs in the hypothalamus, but whether astrocytes participate in this action is unknown. Here we report that astrocyte structural proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, are induced and astrocyte morphology modified by chronic leptin administration (intracerebroventricular, 2 wk), with these changes being inversely related to modifications in synaptic protein densities. Similar changes in glial structural proteins were observed in adult male rats that had increased body weight and circulating leptin levels due to neonatal overnutrition (overnutrition: four pups/litter vs. control: 12 pups/litter). However, acute leptin treatment reduced hypothalamic GFAP levels and induced synaptic protein levels 1 h after administration, with no effect on vimentin. In primary hypothalamic astrocyte cultures leptin also reduced GFAP levels at 1 h, with an induction at 24 h, indicating a possible direct effect of leptin. Hence, one mechanism by which leptin may affect metabolism is by modifying hypothalamic astrocyte morphology, which in turn could alter synaptic inputs to hypothalamic neurons. Furthermore, the responses to acute and chronic leptin exposure are inverse, raising the possibility that increased glial activation in response to chronic leptin exposure could be involved in central leptin resistance. PMID:21343257

  15. 24h Urinary Protein Levels and Urine Protein/Creatinine Ratios Could Probably Forecast the Pathological Classification of HSPN

    PubMed Central

    Ye, Qing; Shang, Shi-qiang; Liu, Ai-min; Zhang, Ting; Shen, Hong-qiang; Chen, Xue-jun; Mao, Jian-hua

    2015-01-01

    This study aimed to assess the relevance of laboratory tests in Henoch-Schönlein purpura nephritis (HSPN) classification, and determine accurate classification factors. This prospective study included 694 HSPN patients who underwent ultrasound-guided percutaneous renal biopsy (PRB). Renal specimens were scored according to International Study of Kidney Disease in Children (ISKDC) classification. Meanwhile, blood samples were immediately collected for laboratory examination. The associations between laboratory parameters and HSPN classification were assessed. Significant differences in levels of serum Th1/Th2 cytokines, immunoglobulins, T-lymphocyte subsets, complement, and coagulation markers were obtained between HSPN patients and healthy children. Interestingly, 24h urinary protein (24h-UPRO) levels and urine protein/urine creatinine ratios could determine HPSN grade IIb, IIIa, and IIIb incidences, with areas under ROC curve of 0.767 and 0.731, respectively. At 24h-UPRO >580.35mg/L, prediction sensitivity and specificity were 75.2% and 70.0%, respectively. These values became 53.0% and 82.3%, respectively, with 24h-UPRO exceeding 1006.25mg/L. At urine protein/urine creatinine > 0.97, prediction sensitivity and specificity were 65.5% and 67.2%, respectively, values that became 57.4% and 80.0%, respectively, at ratios exceeding 1.2. Cell and humoral immunity, coagulation and fibrinolytic systems are all involved in the pathogenesis of HSPN, and type I hypersensitivity may be the disease trigger of HSPN. 24h-UPRO levels and urine protein/creatinine ratios could probably forecast the pathological classification of HSPN. PMID:25996387

  16. Physiological enzymology: The next frontier in understanding protein structure and function at the cellular level.

    PubMed

    Lee, Irene; Berdis, Anthony J

    2016-01-01

    Historically, the study of proteins has relied heavily on characterizing the activity of a single purified protein isolated from other cellular components. This classic approach allowed scientists to unambiguously define the intrinsic kinetic and chemical properties of that protein. The ultimate hope was to extrapolate this information toward understanding how the enzyme or receptor behaves within its native cellular context. These types of detailed in vitro analyses were necessary to reduce the innate complexities of measuring the singular activity and biochemical properties of a specific enzyme without interference from other enzymes and potential competing substrates. However, recent developments in fields encompassing cell biology, molecular imaging, and chemical biology now provide the unique chemical tools and instrumentation to study protein structure, function, and regulation in their native cellular environment. These advancements provide the foundation for a new field, coined physiological enzymology, which quantifies the function and regulation of enzymes and proteins at the cellular level. In this Special Edition, we explore the area of Physiological Enzymology and Protein Function through a series of review articles that focus on the tools and techniques used to measure the cellular activity of proteins inside living cells. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

  17. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae

    PubMed Central

    Masser, Anna E.; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan

    2016-01-01

    Abstract 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 < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat‐shock promoter (PCYC1–HSE), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C‐terminus of a temperature‐sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:26860732

  18. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae.

    PubMed

    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 < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE ), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd.

  19. Ground level environmental protein concentrations in various ecuadorian environments: potential uses of aerosolized protein for ecological research

    USGS Publications Warehouse

    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. Determination of protein levels in soy and peanut oils by colorimetric assay and ELISA.

    PubMed

    Jablonski, Joseph E; Fu, Tong-Jen; Jackson, Lauren S; Gendel, Steven M

    2010-01-01

    Analytical methods are needed for measuring the levels of protein from allergenic food transferred into cooking oil. A simple method for determination of total protein in cooking oils was developed. Oil was extracted with phosphate-buffered saline with 0.05% Tween (PBST) and the extracts were partitioned with hexane to remove residual oil. Total protein in the PBST extracts was assayed with bicinchoninic acid (BCA), micro-BCA, reducing-agent compatible BCA and CB-XT kits. These methods were used to measure recovery of protein from peanut butter spikes of soy and peanut oil in the range of 50-1000 ppm. Recoveries were generally above 70%. However, the BCA and micro-BCA assays were subject to interference and enhanced color formation which were probably due to co-extracted antioxidants present in oil. The reducing agent-compatible BCA and CB-X protein assays reduced interference and gave lower protein values in crude, cold-pressed, and refined peanut oils. Heating oil to 180 degrees C before extraction also reduced interference-induced color enhancement. A commercial ELISA test kit was also used to measure peanut protein in oil spiked with peanut butter. Recovery of peanut residues measured by ELISA was significantly decreased when the peanut butter-spiked oil was heated to 180 degrees C compared to unheated oil. Recovery of spiked peanut butter protein measured by the buffer extraction-colorimetric method was not decreased in heated oil. The method developed here could be used to determine protein levels in crude and refined oil, and to assess the potential for allergen cross-contact from reused cooking oil.

  1. mRNA and Protein levels of rat pancreas specific protein disulphide isomerase are downregulated during Hyperglycemia.

    PubMed

    Gupta, Rajani; Bhar, Kaushik; Sen, Nandini; Bhowmick, Debajit; Mukhopadhyay, Satinath; Panda, Koustubh; Siddhanta, Anirban

    2016-02-01

    Diabetes (Type I and Type II) which affects nearly every organ in the body is a multi-factorial non-communicable disorder. Hyperglycemia is the most characteristic feature of this disease. Loss of beta cells is common in both types of diabetes whose detailed cellular and molecular mechanisms are yet to be elucidated. As this disease is complex, identification of specific biomarkers for its early detection, management and devising new therapies is challenging. Based on the fact that functionally defective proteins provide the biochemical basis for many diseases, in this study, we tried to identify differentially expressed proteins during hyperglycemia. For that, hyperglycemia was induced in overnight fasted rats by intra-peritoneal injection of streptozotocin (STZ). The pancreas was isolated from control and treated rats for subsequent analyses. The 2D-gel electrophoresis followed by MALDI-TOF-MS-MS analyses revealed several up- and down-regulated proteins in hyperglycemic rat pancreas including the downregulation of a pancreas specific isoform of protein disulphide isomerase a2 (Pdia2).This observation was validated by western blot. Quantitative PCR experiments showed that the level of Pdia2 mRNA is also proportionally reduced in hyperglycemic pancreas.

  2. Flour sodium dodecyl sulfate (SDS)-extractable protein level as a cookie flour quality indicator.

    PubMed

    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.

  3. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    PubMed

    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.

  4. Effects of cell-cycle-dependent expression on random fluctuations in protein levels

    PubMed Central

    Soltani, Mohammad

    2016-01-01

    Expression of many genes varies as a cell transitions through different cell-cycle stages. How coupling between stochastic expression and cell cycle impacts cell-to-cell variability (noise) in the level of protein is not well understood. We analyse a model where a stable protein is synthesized in random bursts, and the frequency with which bursts occur varies within the cell cycle. Formulae quantifying the extent of fluctuations in the protein copy number are derived and decomposed into components arising from the cell cycle and stochastic processes. The latter stochastic component represents contributions from bursty expression and errors incurred during partitioning of molecules between daughter cells. These formulae reveal an interesting trade-off: cell-cycle dependencies that amplify the noise contribution from bursty expression also attenuate the contribution from partitioning errors. We investigate the existence of optimum strategies for coupling expression to the cell cycle that minimize the stochastic component. Intriguingly, results show that a zero production rate throughout the cell cycle, with expression only occurring just before cell division, minimizes noise from bursty expression for a fixed mean protein level. By contrast, the optimal strategy in the case of partitioning errors is to make the protein just after cell division. We provide examples of regulatory proteins that are expressed only towards the end of the cell cycle, and argue that such strategies enhance robustness of cell-cycle decisions to the intrinsic stochasticity of gene expression. PMID:28083102

  5. Effects of cell-cycle-dependent expression on random fluctuations in protein levels.

    PubMed

    Soltani, Mohammad; Singh, Abhyudai

    2016-12-01

    Expression of many genes varies as a cell transitions through different cell-cycle stages. How coupling between stochastic expression and cell cycle impacts cell-to-cell variability (noise) in the level of protein is not well understood. We analyse a model where a stable protein is synthesized in random bursts, and the frequency with which bursts occur varies within the cell cycle. Formulae quantifying the extent of fluctuations in the protein copy number are derived and decomposed into components arising from the cell cycle and stochastic processes. The latter stochastic component represents contributions from bursty expression and errors incurred during partitioning of molecules between daughter cells. These formulae reveal an interesting trade-off: cell-cycle dependencies that amplify the noise contribution from bursty expression also attenuate the contribution from partitioning errors. We investigate the existence of optimum strategies for coupling expression to the cell cycle that minimize the stochastic component. Intriguingly, results show that a zero production rate throughout the cell cycle, with expression only occurring just before cell division, minimizes noise from bursty expression for a fixed mean protein level. By contrast, the optimal strategy in the case of partitioning errors is to make the protein just after cell division. We provide examples of regulatory proteins that are expressed only towards the end of the cell cycle, and argue that such strategies enhance robustness of cell-cycle decisions to the intrinsic stochasticity of gene expression.

  6. Retinol-binding protein-4 and hs-CRP levels in patients with migraine.

    PubMed

    Tanik, Nermin; Celikbilek, Asuman; Metin, Aslı; Gocmen, Ayse Yesim; Inan, Levent Ertugrul

    2015-10-01

    Retinol-binding protein-4 (RBP4) and high-sensitivity C-reactive protein (hs-CRP) levels are associated with inflammation in patients with migraine. The release of proinflammatory cytokines during migraine results in recurrent sterile neurogenic inflammation. This study aimed to determine the correlation between RBP4 and hs-CRP levels, and migraine, which is considered an inflammatory disease. The study included 48 migraine patients and 40 age- and gender-matched controls. Migraine was diagnosed according to International Classification of Headache Disorders-II. The serum RBP4 level was measured using a commercial ELISA kit and hs-CRP was measured using an enzyme immunoassay test kit. The serum RBP4 level was significantly lower in the migraine patients than in the controls (P < 0.001), whereas the hs-CRP level was significantly higher in the migraine patients (P < 0.001). RBP4 and hs-CRP levels did not differ between the migraine patients with and without aura (P > 0.05). Migraine headache severity, frequency and duration were not correlated with serum RBP or hs-CRP levels (P > 0.05). The observed high hs-CRP level and low RBP4 level in migraine patients suggest that vitamin A might play a major role in the pathogenesis of migraine. It is known that inflammation is a key factor in many diseases. Additional research might result in a better understanding of the anti-inflammatory effects of vitamin A.

  7. Effects of nutritional supplementation on periodontal parameters, carotenoid antioxidant levels, and serum C-reactive protein.

    PubMed

    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.

  8. Protein Fibrillar Nanopolymers: Molecular-Level Insights into Their Structural, Physical and Mechanical Properties

    NASA Astrophysics Data System (ADS)

    Trusova, Valeriya M.

    2015-09-01

    Amyloid fibrils represent a generic class of mechanically strong and stable biomaterials with extremely advantageous properties. Although amyloids were initially associated only with severe neurological disorders, the role of these structures nowadays is shifting from health debilitating to highly beneficial both in biomedical and technological aspects. Intensive involvement of fibrillar assemblies into the wide range of pathogenic and functional processes strongly necessitate the molecular level characterization of the structural, physical and elastic features of protein nanofibrils. In the present contribution, we made an attempt to highlight the up-to-date progress in the understanding of amyloid properties from the polymer physics standpoint. The fundamental insights into protein fibril behavior are essential not only for development of therapeutic strategies to combat the protein misfolding disorders but also for rational and precise design of novel biodegradable protein-based nanopolymers.

  9. Protein phylogenies robustly resolve the deep-level relationships within Euglenozoa.

    PubMed

    Simpson, Alastair G B; Roger, Andrew J

    2004-01-01

    The deepest-level relationships amongst Euglenozoa remain poorly resolved, despite a rich history of morphological examination and numerous molecular phylogenetic studies of small subunit ribosomal RNA (SSU rRNA) data. We address this question using two nuclear-encoded proteins, the cytosolic isoforms of heat shock protein 90 (hsp90) and heat shock protein 70 (hsp70). For both proteins we examined sequences from the three primary groups within Euglenozoa (euglenids, diplonemids, and kinetoplastids), and from their close relatives, Heterolobosea. Maximum likelihood (ML) and ML distance analyses of these proteins support a close relationship between diplonemids and kinetoplastids to the exclusion of the euglenid Euglena gracilis. In hsp90 and combined protein analyses bootstrap support is very strong and alternative topologies are generally rejected by 'approximately unbiased' (AU) tests. This result is consistent with recent molecular biological and morphological data, but contradicts early structural accounts and many SSU rRNA analyses that favour a closer relationship between diplonemids and euglenids. However, a re-examination of an important SSU rRNA data set highlights the instability of the inferences from this marker. The protein analyses also suggest that bodonids are paraphyletic, with trypanosomatids grouping with 'clade 2' and 'clade 3' bodonids to the exclusion of 'clade 1' bodonids.

  10. Effects of prebiotic, protein level, and stocking density on performance, immunity, and stress indicators of broilers.

    PubMed

    Houshmand, M; Azhar, K; Zulkifli, I; Bejo, M H; Kamyab, A

    2012-02-01

    An experiment was conducted to determine the effects of period on the performance, immunity, and some stress indicators of broilers fed 2 levels of protein and stocked at a normal or high stocking density. Experimental treatments consisted of a 2 × 2 × 2 factorial arrangement with 2 levels of prebiotic (with or without prebiotic), 2 levels of dietary CP [NRC-recommended or low CP level (85% of NRC-recommended level)], and 2 levels of stocking density (10 birds/m(2) as the normal density or 16 birds/m(2) as the high density), for a total of 8 treatments. Each treatment had 5 replicates (cages). Birds were reared in 3-tiered battery cages with wire floors in an open-sided housing system under natural tropical conditions. Housing and general management practices were similar for all treatment groups. Starter and finisher diets in mash form were fed from 1 to 21 d and 22 to 42 d of age, respectively. Supplementation with a prebiotic had no significant effect on performance, immunity, and stress indicators (blood glucose, cholesterol, corticosterone, and heterophil:lymphocyte ratio). Protein level significantly influenced broiler performance but did not affect immunity or stress indicators (except for cholesterol level). The normal stocking density resulted in better FCR and also higher antibody titer against Newcastle disease compared with the high stocking density. However, density had no significant effect on blood levels of glucose, cholesterol, corticosterone, and the heterophil:lymphocyte ratio. Significant interactions between protein level and stocking density were observed for BW gain and final BW. The results indicated that, under the conditions of this experiment, dietary addition of a prebiotic had no significant effect on the performance, immunity, and stress indicators of broilers.

  11. Systemic Glucose Level Changes with a Carbohydrate-Restricted and Higher Protein Diet Combined with Exercise

    ERIC Educational Resources Information Center

    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…

  12. Low levels of aflatoxin B1, ricin and milk enhance recombinant protein production in mammalian cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Changing the optimal tissue culture medium by adding low levels of environmental stress such as 1 µM of the fungal toxin aflatoxin B1 (AFB1), 1 ng of the castor bean protein toxin ricin in transduced mammalian cells or 1% reconstituted milk enhances transcription and increases production of the foll...

  13. A simple atomic-level hydrophobicity scale reveals protein interfacial structure.

    PubMed

    Kapcha, Lauren H; Rossky, Peter J

    2014-01-23

    Many amino acid residue hydrophobicity scales have been created in an effort to better understand and rapidly characterize water-protein interactions based only on protein structure and sequence. There is surprisingly low consistency in the ranking of residue hydrophobicity between scales, and their ability to provide insightful characterization varies substantially across subject proteins. All current scales characterize hydrophobicity based on entire amino acid residue units. We introduce a simple binary but atomic-level hydrophobicity scale that allows for the classification of polar and non-polar moieties within single residues, including backbone atoms. This simple scale is first shown to capture the anticipated hydrophobic character for those whole residues that align in classification among most scales. Examination of a set of protein binding interfaces establishes good agreement between residue-based and atomic-level descriptions of hydrophobicity for five residues, while the remaining residues produce discrepancies. We then show that the atomistic scale properly classifies the hydrophobicity of functionally important regions where residue-based scales fail. To illustrate the utility of the new approach, we show that the atomic-level scale rationalizes the hydration of two hydrophobic pockets and the presence of a void in a third pocket within a single protein and that it appropriately classifies all of the functionally important hydrophilic sites within two otherwise hydrophobic pores. We suggest that an atomic level of detail is, in general, necessary for the reliable depiction of hydrophobicity for all protein surfaces. The present formulation can be implemented simply in a manner no more complex than current residue-based approaches.

  14. The Protein-disulfide Isomerase ERp57 Regulates the Steady-state Levels of the Prion Protein*

    PubMed Central

    Torres, Mauricio; Medinas, Danilo B.; Matamala, José Manuel; Woehlbier, Ute; Cornejo, Víctor Hugo; Solda, Tatiana; Andreu, Catherine; Rozas, Pablo; Matus, Soledad; Muñoz, Natalia; Vergara, Carmen; Cartier, Luis; Soto, Claudio; Molinari, Maurizio; Hetz, Claudio

    2015-01-01

    Although the accumulation of a misfolded and protease-resistant form of the prion protein (PrP) is a key event in prion pathogenesis, the cellular factors involved in its folding and quality control are poorly understood. PrP is a glycosylated and disulfide-bonded protein synthesized at the endoplasmic reticulum (ER). The ER foldase ERp57 (also known as Grp58) is highly expressed in the brain of sporadic and infectious forms of prion-related disorders. ERp57 is a disulfide isomerase involved in the folding of a subset of glycoproteins in the ER as part of the calnexin/calreticulin cycle. Here, we show that levels of ERp57 increase mainly in neurons of Creutzfeldt-Jacob patients. Using gain- and loss-of-function approaches in cell culture, we demonstrate that ERp57 expression controls the maturation and total levels of wild-type PrP and mutant forms associated with human disease. In addition, we found that PrP physically interacts with ERp57, and also with the closest family member PDIA1, but not ERp72. Furthermore, we generated a conditional knock-out mouse for ERp57 in the nervous system and detected a reduction in the steady-state levels of the mono- and nonglycosylated forms of PrP in the brain. In contrast, ERp57 transgenic mice showed increased levels of endogenous PrP. Unexpectedly, ERp57 expression did not affect the susceptibility of cells to ER stress in vitro and in vivo. This study identifies ERp57 as a new modulator of PrP levels and may help with understanding the consequences of ERp57 up-regulation observed in human disease. PMID:26170458

  15. [Plasma concentration of C-reactive protein in patients with high estrogen levels].

    PubMed

    Ricoux, R; Pontet, M; Tresca, J P; Engler, R

    1994-01-01

    The monitoring of inflammatory activity in patients with a high level of estrogen is controversial because the significance of a raised estradiol level on C-reactive protein (CRP) concentrations is a debated question. This prompted us to assay CRP by a sensitive Elisa in a sample of 30 patients with ovarian stimulation for in vitro fertilization, thus with high levels of estradiol. For 15 of these women, six to nine plasma samples were analyzed allowing a kinetic study of plasma levels of CRP, estradiol and sex steroid-binding plasma protein (SBP). No significant correlation was found between the concentrations of estradiol and CRP for the 30 patients. In the kinetic study, as mean estradiol levels rose exponentially from 50 to 1400 ng/l between day 5 and 14, the CRP level tended to vary markedly from one patient to another and sometimes from day to day, but there was never any relation with estradiol level. Furthermore, CRP did not significantly modify the slope of the regression line between estradiol concentration and the day of the menstrual cycle. In contrast, the effect of estradiol on SBP was clear, which supports the absence of estradiol effect on CRP level.

  16. Effect of saliva contamination on induced sputum cell counts, IL-8 and eosinophil cationic protein levels.

    PubMed

    Simpson, J L; Timmins, N L; Fakes, K; Talbot, P I; Gibson, P G

    2004-05-01

    Excessive salivary contamination of induced sputum samples prevents the satisfactory examination of lower airway inflammation. The effects of salivary contamination on different sputum fluid phase measures and the levels of salivary contamination preventing analysis are not defined. The present study sought to examine this by investigating the effect of increasing salivary contamination on induced sputum samples. Sputum and saliva samples from subjects with asthma and healthy controls were collected, and treated with dithiothreitol (DTT). Saliva was then added to aliquots of dispersed sputum in increasing proportions (0% to 100%). The effect of increasing saliva contamination was assessed on sputum total cell count, viability, differential cell count and fluid phase levels of interleukin (IL)-8, eosinophil cationic protein (ECP) and total protein. The addition of saliva to induced sputum reduced total cell counts and absolute cell counts but did not change the differential cell count. Levels of fluid phase ECP and IL-8 were significantly reduced with increased salivary contamination. There was a progressive reduction in ECP and IL-8, which reached significance at 70% and 80% saliva contamination, respectively. IL-8 levels corrected for total protein showed no change with increasing saliva concentrations. Induced sputum differential cell counts expressed as the proportion of nonsquamous cells are robust measures that are not influenced by salivary contamination. Studies reporting total and absolute cell counts and fluid phase mediator levels require control for squamous contamination.

  17. Association of MASP2 polymorphisms and protein levels with rheumatic fever and rheumatic heart disease.

    PubMed

    Catarino, Sandra Jeremias dos Santos; Boldt, Angelica Beate Winter; Beltrame, Marcia Holsbach; Nisihara, Renato Mitsunori; Schafranski, Marcelo Derbli; de Messias-Reason, Iara Jose

    2014-12-01

    MASP-2 is a key protein of the lectin pathway of complement system. Several MASP2 polymorphisms were associated with MASP-2 serum levels or functional activity. Here we investigated a possible association between MASP2 polymorphisms and MASP-2 serum levels with the susceptibility to rheumatic fever (RF) and rheumatic heart disease (RHD). We haplotyped 11 MASP2 polymorphisms with multiplex sequence-specific PCR in 145 patients with history of RF from south Brazil (103 with RHD and 42 without cardiac lesion [RFo]) and 342 healthy controls. MASP-2 levels were determined by ELISA. The low MASP-2 producing p.377A and p.439H variants were negatively associated with RF (P=0.02, OR=0.36) and RHD (P=0.01, OR=0.25). In contrast, haplotypes that share the intron 9 - exon 12 g.1961795C, p.371D, p.377V and p.439R polymorphisms increased the susceptibility to RHD (P=0.02, OR=4.9). MASP-2 levels were associated with MASP2 haplotypes and were lower in patients (P<0.0001), which may reflect protein consumption due to complement activation. MASP2 gene polymorphisms and protein levels seem to play an important role in the development of RF and establishment of RHD.

  18. Understanding epigenetic regulation: Tracking protein levels across multiple generations of cells

    NASA Astrophysics Data System (ADS)

    Rowat, A. C.; Weitz, D. A.

    2009-11-01

    Cells and organisms are remarkably robust: they alter the variety and levels of expressed genes and proteins in response to environmental stimuli, including temperature, chemicals, and the stiffness of their surroundings. Ultimately changes in gene and protein expression can result in a distinct phenotypic state, which in some cases is maintained over multiple generations; the ability to pass on a particular phenotypic state to progeny cells is critical for differentiation. Moreover, epigenetic regulation of phenotype is also thought to provide an evolutionary advantage for a population of cells adapting to a fluctuating environment on faster timescales than the occurrence of genetic mutations. However, simple methods to study patterns of gene and protein expression on multi-generational timescales are sparse. Here we describe a technique to study lineages of single cells over multiple generations using a microfluidic device; this reveals patterns of expression where protein levels are correlated across multiple generations. Such quantitative information of protein expression in the context of pedigree remains hidden when studying the population as an ensemble.

  19. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

    PubMed Central

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L.

    2015-01-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

  20. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level.

    PubMed

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L

    2015-06-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved--all the homologous exons we identified evolved over 460 million years ago--and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles.

  1. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

    PubMed Central

    Gao, Meili; Li, Yongfei; Xue, Xiaochang; Wang, Xianfeng; Long, Jiangang

    2012-01-01

    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. PMID:23093835

  2. An RBCC protein implicated in maintenance of steady-state neuregulin receptor levels.

    PubMed

    Diamonti, A John; Guy, Pamela M; Ivanof, Caryn; Wong, Karen; Sweeney, Colleen; Carraway, Kermit L

    2002-03-05

    Despite numerous recent advances in our understanding of the molecular mechanisms underlying receptor tyrosine kinase down-regulation and degradation in response to growth factor binding, relatively little is known about ligand-independent receptor tyrosine kinase degradation mechanisms. In a screen for proteins that might regulate the trafficking or localization of the ErbB3 receptor, we have identified a tripartite or RBCC (RING, B-box, coiled-coil) protein that interacts with the cytoplasmic tail of the receptor in an activation-independent manner. We have named this protein Nrdp1 for neuregulin receptor degradation protein-1. Northern blotting reveals ubiquitous distribution of Nrdp1 in human adult tissues, but message is particularly prominent in heart, brain, and skeletal muscle. Nrdp1 interacts specifically with the neuregulin receptors ErbB3 and ErbB4 and not with epidermal growth factor receptor or ErbB2. When coexpressed in COS7 cells, Nrdp1 mediates the redistribution of ErbB3 from the cell surface to intracellular compartments and induces the suppression of ErbB3 and ErbB4 receptor levels but not epidermal growth factor receptor or ErbB2 levels. A putative dominant-negative form of Nrdp1 potentiates neuregulin-stimulated Erk1/2 activity in transfected MCF7 breast tumor cells. Our observations suggest that Nrdp1 may act to regulate steady-state cell surface neuregulin receptor levels, thereby influencing the efficiency of neuregulin signaling.

  3. An RBCC protein implicated in maintenance of steady-state neuregulin receptor levels

    PubMed Central

    Diamonti, A. John; Guy, Pamela M.; Ivanof, Caryn; Wong, Karen; Sweeney, Colleen; Carraway, Kermit L.

    2002-01-01

    Despite numerous recent advances in our understanding of the molecular mechanisms underlying receptor tyrosine kinase down-regulation and degradation in response to growth factor binding, relatively little is known about ligand-independent receptor tyrosine kinase degradation mechanisms. In a screen for proteins that might regulate the trafficking or localization of the ErbB3 receptor, we have identified a tripartite or RBCC (RING, B-box, coiled–coil) protein that interacts with the cytoplasmic tail of the receptor in an activation-independent manner. We have named this protein Nrdp1 for neuregulin receptor degradation protein-1. Northern blotting reveals ubiquitous distribution of Nrdp1 in human adult tissues, but message is particularly prominent in heart, brain, and skeletal muscle. Nrdp1 interacts specifically with the neuregulin receptors ErbB3 and ErbB4 and not with epidermal growth factor receptor or ErbB2. When coexpressed in COS7 cells, Nrdp1 mediates the redistribution of ErbB3 from the cell surface to intracellular compartments and induces the suppression of ErbB3 and ErbB4 receptor levels but not epidermal growth factor receptor or ErbB2 levels. A putative dominant-negative form of Nrdp1 potentiates neuregulin-stimulated Erk1/2 activity in transfected MCF7 breast tumor cells. Our observations suggest that Nrdp1 may act to regulate steady-state cell surface neuregulin receptor levels, thereby influencing the efficiency of neuregulin signaling. PMID:11867753

  4. Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Parekh, R N; Wittrup, K D

    1997-01-01

    The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector. Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion. Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER). Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels. Optimal specific productivity of a standard laboratory strain of S. cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S. cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion. Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture.

  5. Plasma C-Reactive Protein Levels Are Associated With Improved Outcome in ARDS

    PubMed Central

    Bajwa, Ednan K.; Khan, Uzma A.; Januzzi, James L.; Gong, Michelle N.; Thompson, B. Taylor; Christiani, David C.

    2009-01-01

    Background: C-reactive protein (CRP) has been studied as a marker of systemic inflammation and outcome in a number of diseases, but little is known about its characteristics in ARDS. We sought to examine plasma levels of CRP in patients with ARDS and their relationship to outcome and measures of illness severity. Methods: We measured CRP levels in 177 patients within 48 h of disease onset and tested the association of protein level with 60-day mortality, 28-day daily organ dysfunction scores, and number of ventilator-free days. Results: We found that CRP levels were significantly lower in nonsurvivors when compared with survivors (p = 0.02). Mortality rate decreased with increasing CRP decile (p = 0.02). An increasing CRP level was associated with a significantly higher probability of survival at 60 days (p = 0.005). This difference persisted after adjustment for age and severity of illness in a multivariable model (p = 0.009). Multivariable models were also used to show that patients in the group with higher CRP levels had significantly lower organ dysfunction scores (p = 0.001) and more ventilator-free days (p = 0.02). Conclusions: Increasing plasma levels of CRP within 48 h of ARDS onset are associated with improved survival, lower organ failure scores, and fewer days of mechanical ventilation. These data appear to be contrary to the established view that CRP is solely a marker of systemic inflammation. PMID:19411291

  6. Chronic high levels of the RCAN1-1 protein may promote neurodegeneration and Alzheimer disease.

    PubMed

    Ermak, Gennady; Davies, Kelvin J A

    2013-09-01

    The RCAN1 gene encodes three different protein isoforms: RCAN1-4, RCAN1-1L, and RCAN1-1S. RCAN1-1L is the RCAN1 isoform predominantly expressed in human brains. RCAN1 proteins have been shown to regulate various other proteins and cellular functions, including calcineurin, glycogen synthase kinase-3β (GSK-3β), the mitochondrial adenine nucleotide transporter (ANT), stress adaptation, ADP/ATP exchange in mitochondria, and the mitochondrial permeability transition pore (mtPTP). The effects of increased RCAN1 gene expression seem to depend both on the specific RCAN1 protein isoform(s) synthesized and on the length of time the level of each isoform is elevated. Transiently elevated RCAN1-4 and RCAN1-1L protein levels, lasting just a few hours, can be neuroprotective under acute stress conditions, including acute oxidative stress. We propose that, by transiently inhibiting the phosphatase calcineurin, RCAN1-4 and RCAN1-1L may reinforce and extend protective stress-adaptive cell responses. In contrast, prolonged elevation of RCAN1-1L levels is associated with the types of neurodegeneration observed in several diseases, including Alzheimer disease and Down syndrome. RCAN1-1L levels can also be increased by multiple chronic stresses and by glucocorticoids, both of which can cause neurodegeneration. Although increasing levels of RCAN1-1L for just a few months has no overtly obvious neurodegenerative effect, it does suppress neurogenesis. Longer term elevation of RCAN1-1L levels (for at least 16 months), however, can lead to the first signs of neurodegeneration. Such neurodegeneration may be precipitated by (RCAN1-1L-mediated) prolonged calcineurin inhibition and GSK-3β induction/activation, both of which promote tau hyperphosphorylation, and/or by (RCAN1-1L-mediated) effects on the mitochondrial ANT, diminished ATP/ADP ratio, opening of the mtPTP, and mitochondrial autophagy. We propose that RCAN1-1L operates through various molecular mechanisms, primarily dependent upon

  7. Comparison of C-reactive protein and high-sensitivity C-reactive protein levels in patients on hemodialysis.

    PubMed

    Helal, Imed; Zerelli, Lilia; Krid, Madiha; ElYounsi, Fethi; Ben Maiz, Hedi; Zouari, Bechir; Adelmoula, Jaouida; Kheder, Adel

    2012-05-01

    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.

  8. Increased myogenic repressor Id mRNA and protein levels in hindlimb muscles of aged rats.

    PubMed

    Alway, Stephen E; Degens, Hans; Lowe, Dawn A; Krishnamurthy, Gururaj

    2002-02-01

    The objective of this study was to determine if levels of repressors to myogenic regulatory factors (MRFs) differ between muscles from young adult and aged animals. Total RNA from plantaris, gastrocnemius, and soleus muscles of Fischer 344 x Brown Norway rats aged 9 mo (young adult, n = 10) and 37 mo (aged, n = 10) was reverse transcribed and then amplified by PCR. To obtain a semiquantitative measure of the mRNA levels, PCR signals were normalized to cyclophilin or 18S signals from the corresponding reverse transcription product. Normalization to cyclophilin and 18S gave similar results. The mRNA levels of MyoD and myogenin were approximately 275-650% (P < 0.001) and approximately 500-1,100% (P < 0.001) greater, respectively, in muscles from aged compared with young adults. In contrast, the protein levels were lower in plantaris and gastrocnemius muscles and similar in the soleus muscle of aged vs. young adult rats. Id repressor mRNA levels were approximately 300-900% greater in fast and slow muscles of aged animals (P < or = 0.02), and Mist 1 mRNA was approximately 50% greater in the plantaris and gastrocnemius muscles (P < 0.01). The mRNA level of Twist mRNA was not significantly affected by aging. Id-1, Id-2, and Id-3 protein levels were approximately 17-740% greater (P < 0.05) in hindlimb muscles of aged rats compared with young adult rats. The elevated levels of Id mRNA and protein suggest that MRF repressors may play a role in gene regulation of fast and slow muscles in aged rats.

  9. Effects of Heme Proteins on Nitric Oxide Levels and Cell Viability in Isolated PMNs: A Mechanism of Toxicity

    DTIC Science & Technology

    2000-03-01

    study. Therapeutic agents that inhibit heme Fig. 10. Concentration-dependent effect of oxyMb on four apoptotic endpoints in PMA-stimulated PMNs. (A) OxyMb...heme proteins suggests that therapeutic interventions should focus on strategies of both reducing NO· levels in tissues and levels of free heme proteins...from serum by plasmapheresis and binding to haptoglobin, reduce binding of heme proteins to cells that produce oxidants, and oxidizing the heme protein

  10. Tumor redox metabolism correlation with the expression level of red fluorescent protein

    NASA Astrophysics Data System (ADS)

    Sha, Shuang; Wang, Anle; Lin, Qiaoya; Zhang, Zhihong

    2015-03-01

    The redox metabolism is variable and complicated with the progress of tumor development. Whether the tumor redox state will affect the exogenous gene expression or not, are still not clear now . To investigate the relationship between tumor endogenous redox state and the exogenous gene expression level, a far red fluorescent protein fRFP was used to monitor tumor cells proliferation and as an exogenous protein expression in tumors. NADH (nicotinamide adenine dinucleotide) and Fp (flavin protein) are two important coenzymes in the mitochondria respiratory chain, which can be as a standard representation for redox metabolism state. Three tumor subcutaneous models (melanoma, human pancreatic carcinoma and nasopharyngeal carcinoma) were used to observe their redox state and protein expression by our home-made redox scanner. The results showed that the distribution of fRFP fluorescent protein expression in the inner tumor regions are heterogeneous, and the fluorescent intensity of fRFP and the fluorescent intensity of NADH have high correlation. In addition, we also found the linear coefficient in three tumors are different, the value of coefficient is (R2 = 0.966 and R2 = 0.943) in melanoma, (R2 = 0.701 and R2 = 0.942) in human pancreatic carcinoma, and (R2 = 0.994) in nasopharyngeal carcinoma, respectively. From these results, we consider that the exogenous protein expression of fRFP in tumor had some relationship with the tumor redox state of NADH.

  11. High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

    PubMed Central

    Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

    2016-01-01

    Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

  12. Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

    SciTech Connect

    Shi, Tujin; Sun, Xuefei; Gao, Yuqian; Fillmore, Thomas L.; Schepmoes, Athena A.; Zhao, Rui; He, Jintang; Moore, Ronald J.; Kagan, Jacob; Rodland, Karin D.; Liu, Tao; Liu, Alvin Y.; Smith, Richard D.; Tang, Keqi; Camp, David G.; Qian, Weijun

    2013-07-05

    We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50-100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion and the multiplexing potential of this technique. Limits of quantification (LOQs) at low ng/mL levels with a median CV of ~12% were achieved for proteins spiked into human female serum using as little as 2 µL serum. PRISM-SRM provided up to ~1000-fold improvement in the LOQ when compared to conventional SRM measurements. Multiplexing capability of PRISM-SRM was also evaluated by two sets of serum samples with 6 and 21 target peptides spiked at the low attomole/µL levels. The results from SRM measurements for pooled or post-concatenated samples were comparable to those obtained from individual peptide fractions in terms of signal-to-noise ratios and SRM peak area ratios of light to heavy peptides. PRISM-SRM was applied to measure several ng/mL-level endogenous plasma proteins, including prostate-specific antigen, in clinical patient sera where correlation coefficients > 0.99 were observed between the results from PRISM-SRM and ELISA assays. Our results demonstrate that PRISM-SRM can be successfully used for quantification of low-abundance endogenous proteins in highly complex samples. Moderate throughput (50 samples/week) can be achieved by applying the post-concatenation or fraction multiplexing strategies. We anticipate broad applications for targeted PRISM

  13. Coupling between protein level selection and codon usage optimization in the evolution of bacteria and archaea.

    PubMed

    Ran, Wenqi; Kristensen, David M; Koonin, Eugene V

    2014-03-25

    The relationship between the selection affecting codon usage and selection on protein sequences of orthologous genes in diverse groups of bacteria and archaea was examined by using the Alignable Tight Genome Clusters database of prokaryote genomes. The codon usage bias is generally low, with 57.5% of the gene-specific optimal codon frequencies (Fopt) being below 0.55. This apparent weak selection on codon usage contrasts with the strong purifying selection on amino acid sequences, with 65.8% of the gene-specific dN/dS ratios being below 0.1. For most of the genomes compared, a limited but statistically significant negative correlation between Fopt and dN/dS was observed, which is indicative of a link between selection on protein sequence and selection on codon usage. The strength of the coupling between the protein level selection and codon usage bias showed a strong positive correlation with the genomic GC content. Combined with previous observations on the selection for GC-rich codons in bacteria and archaea with GC-rich genomes, these findings suggest that selection for translational fine-tuning could be an important factor in microbial evolution that drives the evolution of genome GC content away from mutational equilibrium. This type of selection is particularly pronounced in slowly evolving, "high-status" genes. A significantly stronger link between the two aspects of selection is observed in free-living bacteria than in parasitic bacteria and in genes encoding metabolic enzymes and transporters than in informational genes. These differences might reflect the special importance of translational fine-tuning for the adaptability of gene expression to environmental changes. The results of this work establish the coupling between protein level selection and selection for translational optimization as a distinct and potentially important factor in microbial evolution. IMPORTANCE Selection affects the evolution of microbial genomes at many levels, including both

  14. Atomic-Level Structure Characterization of an Ultrafast Folding Mini-Protein Denatured State

    PubMed Central

    Rogne, Per; Ozdowy, Przemysław; Richter, Christian; Saxena, Krishna; Schwalbe, Harald; Kuhn, Lars T.

    2012-01-01

    Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing 15N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R2 rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding. PMID:22848459

  15. Altered MCM Protein Levels and Autophagic Flux in Aged and Systemic Sclerosis Dermal Fibroblasts

    PubMed Central

    Dumit, Verónica I.; Küttner, Victoria; Käppler, Jakob; Piera-Velazquez, Sonsoles; Jimenez, Sergio A.; Bruckner-Tuderman, Leena; Uitto, Jouni; Dengjel, Jörn

    2014-01-01

    Aging is a common risk factor of many disorders. With age, the level of insoluble extracellular matrix increases leading to increased stiffness of a number of tissues. Matrix accumulation can also be observed in fibrotic disorders, such as systemic sclerosis (SSc). Although the intrinsic aging process in skin is phenotypically distinct from SSc, here we demonstrate similar behavior of aged and SSc skin fibroblasts in culture. We have used quantitative proteomics to characterize the phenotype of dermal fibroblasts from healthy subjects of various ages and from patients with SSc. Our results demonstrate that proteins involved in DNA and RNA processing decrease with age and in SSc, while those involved in mitochondrial and other metabolic processes behave the opposite. Specifically, mini-chromosome maintenance (MCM) helicase proteins are less abundant with age and SSc, and they exhibit an altered subcellular distribution. We observed that lower levels of MCM7 correlate with reduced cell proliferation, lower autophagic capacity and higher intracellular protein expression phenotypes of aged and SSc cells. Additionally, we show that SSc fibroblasts exhibit higher levels of senescence than their healthy counterparts, suggesting further similarities between the fibrotic disorder and the aging process. Hence, at the molecular level, SSc fibroblasts exhibit intrinsic characteristics of fibroblasts from aged skin. PMID:24496236

  16. Obese patients have higher circulating protein levels of dipeptidyl peptidase IV.

    PubMed

    Stengel, Andreas; Goebel-Stengel, Miriam; Teuffel, Pauline; Hofmann, Tobias; Buße, Petra; Kobelt, Peter; Rose, Matthias; Klapp, Burghard F

    2014-11-01

    Dipeptidyl peptidase IV (DPPIV) is a protease with broad distribution involved in various homeostatic processes such as immune defense, psychoneuroendocrine functions and nutrition. While DPPIV protein levels were investigated in patients with hyporectic disorders, less is known under conditions of obesity. Therefore, we investigated DPPIV across a broad range of body mass index (BMI). Blood samples from hospitalized patients with normal weight (BMI 18.5-25 kg/m(2)), anorexia nervosa (BMI <17.5 kg/m(2)) and obesity (BMI 30-40, 40-50 and >50 kg/m(2), n = 15/group) were tested cross-sectionally and DPPIV concentration and total enzyme activity and the DPPIV targets, pancreatic polypeptide (PP) and glucagon-like peptide (GLP-1) were measured. DPPIV protein expression was detected in human plasma indicated by a strong band at the expected size of 110 kDa and another major band at 50 kDa, likely representing a fragment comprised of two heavy chains. Obese patients had higher DPPIV protein levels compared to normal weight and anorexics (+50%, p<0.05) resulting in a positive correlation with BMI (r = 0.34, p = 0.004). DPPIV serum activity was similar in all groups (p>0.05), while the concentration/activity ratio was higher in obese patients (p<0.05). Plasma PP levels were highest in anorexic patients (∼ 2-fold increase compared to other groups, p<0.05), whereas GLP-1 did not differ among groups (p<0.05). Taken together, circulating DPPIV protein levels depend on body weight with increased levels in obese resulting in an increased concentration/activity ratio. Since DPPIV deactivates food intake-inhibitory hormones like PP, an increased DPPIV concentration/activity ratio might contribute to reduced food intake-inhibitory signaling under conditions of obesity.

  17. Gallium nitrate regulates rat osteoblast expression of osteocalcin protein and mRNA levels.

    PubMed

    Guidon, P T; Salvatori, R; Bockman, R S

    1993-01-01

    Gallium nitrate, a group IIIa metal salt, has been found to be clinically effective for the treatment of accelerated bone resorption in cancer-related hypercalcemia and Paget's disease. Here we report the effects of gallium nitrate on osteocalcin mRNA and protein levels on the rat osteoblast-like cell line ROS 17/2.8. Gallium nitrate reduced both constitutive and vitamin D3-stimulated osteocalcin protein levels in culture medium by one-half and osteocalcin mRNA levels to one-third to one-tenth of control. Gallium nitrate also inhibited vitamin D3 stimulation of osteocalcin and osteopontin mRNA levels but did not affect constitutive osteopontin mRNA levels. Among several different metals examined, gallium was unique in its ability to reduce osteocalcin mRNA levels without decreasing levels of other mRNAs synthesized by ROS 17/2.8 cells. The effects of gallium nitrate on osteocalcin mRNA and protein synthesis mimic those seen when ROS 17/2.8 cells are exposed to transforming growth factor beta 1 (TGF beta 1); however, TGF-beta 1 was not detected in gallium nitrate-treated ROS 17/2.8 cell media. Use of the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that gallium nitrate did not alter the stability of osteocalcin mRNA. Transient transfection assays using the rat osteocalcin promoter linked to the bacterial reporter gene chloramphenicol acetyltransferase indicated that gallium nitrate blocked reporter gene expression stimulated by the osteocalcin promoter. This is the first reported effect of gallium nitrate on isolated osteoblast cells.

  18. Ultrasensitive detection of proteins and sugars at single-cell level

    PubMed Central

    Watabe, Satoshi; Morikawa, Mika; Kaneda, Mugiho; Nakaishi, Kazunari; Nakatsuma, Akira; Ninomiya, Masaki; Yoshimura, Teruki; Miura, Toshiaki; Ito, Etsuro

    2016-01-01

    ABSTRACT Each cell produces its own responses even if it appears identical to other cells. To analyze these individual cell characteristics, we need to measure trace amounts of molecules in a single cell. Nucleic acids in a single cell can be easily amplified by polymerase chain reaction, but single-cell measurement of proteins and sugars will require de novo techniques. In the present study, we outline the techniques we have developed toward this end. For proteins, our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide cycling can detect proteins at subattomoles per assay. For sugars, fluorescence correlation spectroscopy coupled with glucose oxidase-catalyzed reaction allows us to measure glucose at tens of nM. Our methods thus offer versatile techniques for single-cell-level analyses, and they are hoped to strongly promote single-cell biology as well as to develop noninvasive tests in clinical medicine. PMID:27064305

  19. [Oxidative modification of rat blood proteins after destruction capsaicin-sensitive nerve and change of nitric oxide level].

    PubMed

    Tolochko, Z S; Spiridonov, V K

    2010-01-01

    Content of blood protein carbonyl derivates in rats are determined to assess oxidative modification of protein after destruction of capsaicin-sensitive nerve and change of nitric oxide (NO) level. Deafferentation of these nerves produces increase of the protein carbonyl derivates content. The increase of NO by L-arginine does not affect protein oxidative destruction produced by ablation of capsaicin-sensitive nerve. Selective inhibitor of neuronal synthase NO (n-NOS) 7-nitroindazole (7-NI) results in similar effect. L-NAME increased oxidative destruction of proteins. These results demonstrate that deafferentation of capsaicin-sensitive nerve induces oxidative destruction of proteins. NO has party to mediating oxidative modification of proteins.

  20. Heat Stress Decreases Levels of Nutrient-Uptake and -Assimilation Proteins in Tomato Roots

    PubMed Central

    Giri, Anju; Heckathorn, Scott; Mishra, Sasmita; Krause, Charles

    2017-01-01

    Global warming will increase root heat stress, which is already common under certain conditions. Effects of heat stress on root nutrient uptake have rarely been examined in intact plants, but the limited results indicate that heat stress will decrease it; no studies have examined heat-stress effects on the concentration of nutrient-uptake proteins. We grew Solanum lycopersicum (tomato) at 25 °C/20 °C (day/night) and then transferred some plants for six days to 35 °C /30 °C (moderate heat) or 42 °C/37 °C (severe heat) (maximum root temperature = 32 °C or 39 °C, respectively); plants were then moved back to control conditions for seven days to monitor recovery. In a second experiment, plants were grown for 15 days at 28 °C/23 °C, 32 °C/27 °C, 36 °C/31 °C, and 40 °C/35 °C (day/night). Concentrations of nutrient-uptake and -assimilation proteins in roots were determined using protein-specific antibodies and ELISA (enzyme-linked immunosorbent assay). In general, (1) roots were affected by heat more than shoots, as indicated by decreased root:shoot mass ratio, shoot vs. root %N and C, and the level of nutrient metabolism proteins vs. less sensitive photosynthesis and stomatal conductance; and (2) negative effects on roots were large and slow-to-recover only with severe heat stress (40 °C–42 °C). Thus, short-term heat stress, if severe, can decrease total protein concentration and levels of nutrient-uptake and -assimilation proteins in roots. Hence, increases in heat stress with global warming may decrease crop production, as well as nutritional quality, partly via effects on root nutrient relations. PMID:28106834

  1. Levels of metacaspase1 and chaperones related to protein quality control in alcoholic and nonalcoholic steatohepatitis.

    PubMed

    Mendoza, Alejandro S; Dorce, Jacques; Peng, Yue; French, Barbara A; Tillman, Brittany; Li, Jun; French, Samuel W

    2015-02-01

    Efficient management of misfolded or aggregated proteins in ASH and NASH is crucial for continued hepatic viability. Cellular protein quality control systems play an important role in the pathogenesis and progression of ASH and NASH. In a recent study, elevated Mca1 expression counteracted aggregation and accumulation of misfolded proteins and extended the life span of the yeast Saccharomyces cerevisiae (Hill et al, 2014). Mca1 may also associate with Ssa1 and Hsp104 in disaggregation and fragmentation of aggregated proteins and their subsequent degradation through the ER-associated degradation (ERAD) pathway. If degradation is not available, protection of the cellular environment from a misfolded protein is accomplished by its sequestration into two distinct inclusion bodies (Kaganovich et al., 2008) called the JUNQ (JUxta Nuclear Quality control compartment) and the IPOD (Insoluble Protein Deposit). Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 all play important roles in protein quality control systems. This study aims to measure the expression of Mca1 and related chaperones involved in protein quality control in alcoholic steatohepatitis (ASH), and nonalcoholic steatohepatitis (NASH) compared with normal control liver biopsies. Mca1, Hsp104, Hsp40, Ydj1, Ssa1, VCP/p97, and p62 expressions were measured in three to six formalin-fixed paraffin embedded ASH and NASH liver biopsies and control normal liver specimens by immunofluorescence staining and quantified by immunofluorescence intensity. Mca1, Hsp104, Ydj1 and p62 were significantly upregulated compared to control (p<0.05) in ASH specimens. Hsp40 and VCP/p97 were also uptrending in ASH. In NASH, the only significant difference was the increased expression of Hsp104 compared to control (p<0.05). Ssa1 levels were uptrending in both ASH and NASH specimens. The upregulation of Mca1, Hsp104, Ydj1 and p62 in ASH may be elicited as a response to the chronic exposure of the hepatocytes to the toxicity of alcohol

  2. Association of androgen with gender difference in serum adipocyte fatty acid binding protein levels

    PubMed Central

    Hu, Xiang; Ma, Xiaojing; Pan, Xiaoping; Luo, Yuqi; Xu, Yiting; Xiong, Qin; Bao, Yuqian; Jia, Weiping

    2016-01-01

    Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m2; all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels. PMID:27270834

  3. Ultraviolet-ozone treatment reduces levels of disease-associated prion protein and prion infectivity

    USGS Publications Warehouse

    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.

  4. Warfarin-Induced Skin Necrosis in Patients With Low Protein C Levels.

    PubMed

    Marčić, Marino; Marčić, Ljiljana; Titlić, Marina

    2016-08-01

    Warfarin-induced skin necrosis (WISN) is a rare complication of anticoagulant therapy associated with a high incidence of  morbidity and mortality requiring immediate drug cessation. At particular risk are those with various thrombophilic abnormalities, especially when warfarinisation is undertaken rapidly with large loading doses of warfarin. Cutaneous findings include petechiae that progress to ecchymosis and hemorrhagic bullae. With the increasing number of patients anticoagulated as out-patients for thromboprophylaxis, we are concerned that the incidence of skin necrosis may increase. We present a case of WISN with low protein C level. He was a 50-year-old male who came to our department because of acute infarction in irrigation area of the superior cerebellar artery. He had intermittent atrial fibrillation and was started on anticoagulant therapy.  After few day of therapy, he developed skin necrosis, and his level of protein C was low. Warfarin-induced skin necrosis is a rare but serious complication that can be prevented by routine screening for protein C, protein S or antithrombin deficiencies or for the presence of antiphospholipid antibodies before beginning warfarin therapy.

  5. Phylogenomic evaluation of members above the species level within the phylum Firmicutes based on conserved proteins.

    PubMed

    Zhang, Weiwei; Lu, Zhitang

    2015-04-01

    Currently, numerous taxonomic units above species level of the phylum Firmicutes are ambiguously placed in the phylogeny determined by 16S rRNA gene. Here, we evaluated the use of 16S rRNA gene compared with 81 conserved proteins (CPs) or 41 ribosomal proteins (RPs) as phylogenetic markers and applied this to the analysis of the phylum Firmicutes. Results show that the phylogenetic trees constructed are in good agreement with each other; however, the protein-based trees are able to resolve the relationships between several branches where so far only ambiguous classifications are possible. Thus, the phylogeny deduced based on concatenated proteins provides significant basis for re-classifying members in this phylum. It indicates that the genera Coprothermobacter and Thermodesulfobium represent two new phyla; the families Paenibacillaceae and Alicyclobacillaceae should be elevated to order level; and the families Bacillaceae and Thermodesulfobiaceae should be separated to 2 and 3 families respectively. We also suggest that four novel families should be proposed in the orders Clostridiales and Bacillales, and 11 genera should be moved to other existing families different from the current classification status. Moreover, notably, RPs are a well-suited subset of CPs that could be applied to Firmicutes phylogenetic analysis instead of the 16S rRNA gene.

  6. Daily fluctuations of haematology and blood biochemistry in horses fed varying levels of protein.

    PubMed

    Greppi, G F; Casini, L; Gatta, D; Orlandi, M; Pasquini, M

    1996-09-01

    Changes in the plasma biochemistry of 12 stallions were studied over seven 24 h periods. Twelve healthy male horses of 4 different breeds (Thoroughbred [TB], Anglo-Arabo-Sardo [AAS], Avelignese [Av] and Maremmano [M]) were divided into 3 experimental groups and fed with an isoenergetic maintenance ration with different protein levels. The same amount of food was given daily at 0730 and 1930 h. After 20 days of pretrial, a series of 6 blood samples were collected daily from the jugular vein every 4 h for 7 days. The samples were analysed for packed cell volume (PCV), haemoglobin, plasma glucose, free fatty acids, cholesterol, total plasma protein, urea, creatinine, calcium, phosphorus and enzymatic activities: aspartate aminotransferase (AST), alanine aminotrasferase (ALT), creatine kinase (CK) and lactate dehydrogenase (LDH). The data were analysed following the armonic analysis (Fourier) in order to define the pattern of biorhythm. Many parameters showed a biorhythmical pattern. Protein levels of the diet failed to influence haematology, blood biochemistry and biorhythm. Thoroughbreds showed the highest values of PCV, haemoglobin and creatinine and the lowest total protein, phosphorus and enzymatic activities.

  7. Quantitation of tyrosine hydroxylase, protein levels: Spot immunolabeling with an affinity-purified antibody

    SciTech Connect

    Haycock, J.W. )

    1989-09-01

    Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and {sup 125}I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background {sup 125}I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

  8. Patients with nephrolithiasis had lower fetuin-A protein level in urine and renal tissue.

    PubMed

    Wu, Yong Xian; Li, Cheng Yang; Deng, Yao Liang

    2014-02-01

    Fetuin-A acts as an inhibitor of systemic and local ectopic calcification and inflammatory response, but the role of fetuin-A in the etiology of urolithiasis is still unclear. We aim to investigate the expression of fetuin-A in the serum, urine and renal tissue of patients with or without nephrolithiasis. 48 patients with nephrolithiasis (group A) and 32 individuals without urolithiasis (group B, control group) were enrolled into our study. Level of fetuin-A in serum and urine was measured by ELISA, and expression of fetuin-A in renal tissue was localized and assessed by immunohistochemistry, real-time polymerase chain reaction, and Western blotting, respectively. Indexes of oxidative stress in kidney were evaluated. Other routine serum and urine chemistries for inpatients were measured biochemically. The results showed that fetuin-A expressed widely in the proximal and distal renal tubule, the thin segment of Henle's loop and the collecting duct epithelium. There were no differences in serum fetuin-A level between the two groups. Compared with control group, cellular expression of P47phox and fetuin-A mRNAs in the renal tissue of patients with nephrolithiasis increased, the level of MDA in renal tissue and the level of urinary calcium also increased, but urinary and renal fetuin-A protein and the activities of SOD in renal tissue decreased. Correlation analysis showed that there was a negative correlation between the level of renal fetuin-A protein and the expression of P47phox mRNA and MDA. These results revealed that nephrolithiasis patients had lower fetuin-A protein level in urine and renal tissue.

  9. Calculation of hydrodynamic properties of globular proteins from their atomic-level structure.

    PubMed Central

    García De La Torre, J; Huertas, M L; Carrasco, B

    2000-01-01

    The solution properties, including hydrodynamic quantities and the radius of gyration, of globular proteins are calculated from their detailed, atomic-level structure, using bead-modeling methodologies described in our previous article (, Biophys. J. 76:3044-3057). We review how this goal has been pursued by other authors in the past. Our procedure starts from a list of atomic coordinates, from which we build a primary hydrodynamic model by replacing nonhydrogen atoms with spherical elements of some fixed radius. The resulting particle, consisting of overlapping spheres, is in turn represented by a shell model treated as described in our previous work. We have applied this procedure to a set of 13 proteins. For each protein, the atomic element radius is adjusted, to fit all of the hydrodynamic properties, taking values close to 3 A, with deviations that fall within the error of experimental data. Some differences are found in the atomic element radius found for each protein, which can be explained in terms of protein hydration. A computational shortcut makes the procedure feasible, even in personal computers. All of the model-building and calculations are carried out with a HYDROPRO public-domain computer program. PMID:10653785

  10. The human phenolsulphotransferase polymorphism is determined by the level of expression of the enzyme protein.

    PubMed Central

    Jones, A L; Roberts, R C; Coughtrie, M W

    1993-01-01

    We have examined the expression of platelet phenolsulphotransferase (PST) in 60 individuals. Using an antibody which recognizes both forms of PST present in man (P-PST and M-PST), we determined that the polymorphism of platelet P-PST activity is determined by the level of expression of the enzyme protein. The implications for susceptibility to adverse drug reactions and chemical carcinogenesis are discussed. Images Figure 1 Figure 2 Figure 3 PMID:8257413

  11. Effects of antidepressant drugs on synaptic protein levels and dendritic outgrowth in hippocampal neuronal cultures.

    PubMed

    Seo, Mi Kyoung; Lee, Chan Hong; Cho, Hye Yeon; Lee, Jung Goo; Lee, Bong Ju; Kim, Ji Eun; Seol, Wongi; Kim, Young Hoon; Park, Sung Woo

    2014-04-01

    The alteration of hippocampal plasticity has been proposed to play a critical role in both the pathophysiology and treatment of depression. In this study, the ability of different classes of antidepressant drugs (escitalopram, fluoxetine, paroxetine, sertraline, imipramine, tranylcypromine, and tianeptine) to mediate the expression of synaptic proteins and dendritic outgrowth in rat hippocampal neurons was investigated under toxic conditions induced by B27 deprivation, which causes hippocampal cell death. Postsynaptic density protein-95 (PSD-95), brain-derived neurotrophic factor (BDNF), and synaptophysin (SYP) levels were evaluated using Western blot analyses. Additionally, dendritic outgrowth was examined to determine whether antidepressant drugs affect the dendritic morphology of hippocampal neurons in B27-deprived cultures. Escitalopram, fluoxetine, paroxetine, sertraline, imipramine, tranylcypromine, and tianeptine significantly prevented B27 deprivation-induced decreases in levels of PSD-95, BDNF, and SYP. Moreover, the independent application of fluoxetine, paroxetine, and sertraline significantly increased levels of BDNF under normal conditions. All antidepressant drugs significantly increased the total outgrowth of hippocampal dendrites under B27 deprivation. Specific inhibitors of calcium/calmodulin kinase II (CaMKII), KN-93, protein kinase A (PKA), H-89, or phosphatidylinositol 3-kinase (PI3K), LY294002, significantly decreased the effects of antidepressant drugs on dendritic outgrowth, whereas this effect was observed only with tianeptine for the PI3K inhibitor. Taken together, these results suggest that certain antidepressant drugs can enhance synaptic protein levels and encourage dendritic outgrowth in hippocampal neurons. Furthermore, effects on dendritic outgrowth likely require CaMKII, PKA, or PI3K signaling pathways. The observed effects may be may be due to chronic treatment with antidepressant drugs.

  12. Level of receptor-associated protein moderates cellular susceptibility to pseudomonas exotoxin A.

    PubMed Central

    Mucci, D; Forristal, J; Strickland, D; Morris, R; Fitzgerald, D; Saelinger, C B

    1995-01-01

    Pseudomonas exotoxin A (PE) enters mammalian cells via a receptor-mediated endocytic pathway. The initial step in this pathway is binding to the multiligand receptor termed the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein (LRP). Binding of toxin, and of the many other ligands that bind to LRP, is blocked by the addition of a 39-kDa receptor-associated protein (RAP). Here we show that approximately 40% of the cell-associated LRP is on the surface of toxin-sensitive mouse LM fibroblasts and thus accessible for toxin internalization. The remainder is located intracellularly, primarily in the Golgi region. Mammalian cells exhibit a wide range of sensitivity to PE. To investigate possible reasons for this, we examined the expression levels of both LRP and RAP. Results from a variety of cell lines indicated that there was a positive correlation between LRP expression and toxin sensitivity. In the absence of LRP, cells were as much as 200-fold more resistant to PE compared with sensitive cells. A second group of resistant cells expressed LRP but had a high level of RAP. Thus, a toxin-resistant phenotype would be expected when cells expressed either low levels of LRP or high levels of LRP in the presence of high levels of RAP. We hypothesize that RAP has a pivotal role in moderating cellular susceptibility to PE. PMID:7622212

  13. Birdsong decreases protein levels of FoxP2, a molecule required for human speech.

    PubMed

    Miller, Julie E; Spiteri, Elizabeth; Condro, Michael C; Dosumu-Johnson, Ryan T; Geschwind, Daniel H; White, Stephanie A

    2008-10-01

    Cognitive and motor deficits associated with language and speech are seen in humans harboring FOXP2 mutations. The neural bases for FOXP2 mutation-related deficits are thought to reside in structural abnormalities distributed across systems important for language and motor learning including the cerebral cortex, basal ganglia, and cerebellum. In these brain regions, our prior research showed that FoxP2 mRNA expression patterns are strikingly similar between developing humans and songbirds. Within the songbird brain, this pattern persists throughout life and includes the striatal subregion, Area X, that is dedicated to song development and maintenance. The persistent mRNA expression suggests a role for FoxP2 that extends beyond the formation of vocal learning circuits to their ongoing use. Because FoxP2 is a transcription factor, a role in shaping circuits likely depends on FoxP2 protein levels which might not always parallel mRNA levels. Indeed our current study shows that FoxP2 protein, like its mRNA, is acutely downregulated in mature Area X when adult males sing with some differences. Total corticosterone levels associated with the different behavioral contexts did not vary, indicating that differences in FoxP2 levels are not likely attributable to stress. Our data, together with recent reports on FoxP2's target genes, suggest that lowered FoxP2 levels may allow for expression of genes important for circuit modification and thus vocal variability.

  14. Inclusion of Yucca schidigera extract in diets with different protein levels for dogs.

    PubMed

    Dos Reis, Jéssica S; Zangerônimo, Márcio G; Ogoshi, Rosana C S; França, Janine; Costa, Adriano C; Almeida, Thomás N; Dos Santos, João P F; Pires, Carolina P; Chizzotti, Ana F; Leite, Carlos A L; Saad, Flávia M O B

    2016-08-01

    This study evaluated the effects of inclusion of Yucca schidigera extract (YSE) in two diets with different levels of crude protein (CP) for dogs on facal odour, nutrient digestibility, ammonia concentration in feces and hematological and serum biochemical profiles. Twenty adults Beagles were used, distributed in a randomized block design in a 2 × 4 factorial design (two diets, 25% and 34% CP, and four YSE levels: 0, 250, 500 and 750 mg/kg) with five replicates, obtained during two experimental periods. The fecal odour reduced (P < 0.05) when 500 mg/kg of YSE was used in diets with higher CP. The inclusion of YSE reduced (P < 0.05) fecal ammonia, and the inclusion of 250 and 500 mg/kg YSE reduced intestinal gas. The inclusion of 750 mg/kg YSE increased the mean corpuscular hemoglobin (MCH), alanine aminotransferase (ALT) activity and tended to increase the serum cholesterol concentration, regardless of the protein level of the diets. There was no effect on the digestibility of nutrients, fecal consistency, nitrogen balance and thickness of the intestinal wall. The inclusion of 500 mg/kg YSE is effective in reducing fecal odour in dogs receiving diets with 34% of CP. Regardless of the protein content, YSE reduces fecal ammonia, but may cause adverse effects if included at higher doses.

  15. A Compendium of Caenorhabditis elegans RNA Binding Proteins Predicts Extensive Regulation at Multiple Levels

    PubMed Central

    Tamburino, Alex M.; Ryder, Sean P.; Walhout, Albertha J. M.

    2013-01-01

    Gene expression is regulated at multiple levels, including transcription and translation, as well as mRNA and protein stability. Although systems-level functions of transcription factors and microRNAs are rapidly being characterized, few studies have focused on the posttranscriptional gene regulation by RNA binding proteins (RBPs). RBPs are important to many aspects of gene regulation. Thus, it is essential to know which genes encode RBPs, which RBPs regulate which gene(s), and how RBP genes are themselves regulated. Here we provide a comprehensive compendium of RBPs from the nematode Caenorhabditis elegans (wRBP1.0). We predict that as many as 887 (4.4%) of C. elegans genes may encode RBPs ~250 of which likely function in a gene-specific manner. In addition, we find that RBPs, and most notably gene-specific RBPs, are themselves enriched for binding and modification by regulatory proteins, indicating the potential for extensive regulation of RBPs at many different levels. wRBP1.0 will provide a significant contribution toward the comprehensive delineation of posttranscriptional regulatory networks and will provide a resource for further studies regulation by RBPs. PMID:23390605

  16. Association of HIV Infection and HIV/HCV Coinfection With C-Reactive Protein Levels

    PubMed Central

    Reingold, Jason S.; Wanke, Christine; Kotler, Donald P.; Lewis, Cora E.; Tracy, Russell; Heymsfield, Steven; Tien, Phyllis C.; Bacchetti, Peter; Scherzer, Rebecca; Grunfeld, Carl; Shlipak, Michael G.

    2008-01-01

    Objective Inflammation is a potential mechanism to explain the accelerated atherosclerosis observed in HIV- and hepatitis C virus (HCV)–infected persons. We evaluated C-reactive protein (CRP) in HIV-infected and HIV/HCV-coinfected individuals in the era of effective antiretroviral (ARV) therapy. Design Cross-sectional study of Fat Redistribution and Metabolic Change in HIV Infection (FRAM) cohort and controls from the Coronary Artery Risk Development in Young Adults (CARDIA) study. Methods CRP levels were measured in 1135 HIV-infected participants from the FRAM cohort and 281 controls from the CARDIA study. The associations of HIV and HIV/HCV infection with CRP levels were estimated by multivariable linear regression. Results Compared with controls, HIV monoinfection was associated with an 88% higher CRP level in men (P < 0.0001) but with no difference in women (5%; P = 0.80) in multivariate analysis. CRP levels were not associated with ARV therapy, HIV RNA level, or CD4 cell count. Compared with controls, HIV/HCV coinfection was associated with a 41% lower CRP level in women (P = 0.012) but with no difference in men (+4%; P = 0.90). Among HIV-infected participants, HCV coinfection was associated with 50% lower CRP levels after multivariable analysis (P < 0.0001) in men and women. Greater visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were strongly associated with CRP levels. Among HIV- infected participants, CRP levels were 17% (P < 0.001) and 21% (P = 0.002) higher per doubling of VAT and SAT; among controls, CRP levels were 34% (P < 0.001) and 61% (P = 0.009) higher, respectively. Conclusions In the absence of HCV coinfection, HIV infection is associated with higher CRP levels in men. HCV coinfection is associated with lower CRP levels in men and women. PMID:18344877

  17. Decreased levels of heat shock proteins in gut epithelial cells after exposure to plant lectins

    PubMed Central

    Ovelgonne, J; Koninkx, J; Pusztai, A; Bardocz, S; Kok, W; Ewen, S; Hendriks, H; van Dijk, J E

    2000-01-01

    BACKGROUND—The enterocytes of the intestinal epithelium are regularly exposed to potentially harmful substances of dietary origin, such as lectins. Expression of heat shock proteins (HSPs) by this epithelium may be part of a protective mechanism developed by intestinal epithelial cells to deal with noxious components in the intestinal lumen.
AIM—To investigate if the lectins PHA, a lectin from kidney beans (Phaseolus vulgaris) and WGA, a lectin from wheat germ (Triticum aestivum) could modify the heat shock response in gut epithelial cells and to establish the extent of this effect.
METHODS—Jejunal tissue sections from PHA and WGA fed rats were screened for expression of HSP70, HSP72, and HSP90 using monoclonal antibodies. Differentiated Caco-2 cells, the in vitro counterpart of villus enterocytes, were exposed to 100 µg/ml of PHA-E4 or WGA for 48 hours and investigated for changes in DNA and protein synthesis by double labelling with [2-14C]thymidine and L-[methyl-3H]methionine. The relative concentrations of HSP60, HSP70, HSP72, and HSP90 and binding protein (BiP) in these cells exposed to lectins were analysed by polyacrylamide gel electrophoresis and immunoblotting. To establish if lectin exposed differentiated Caco-2 cells were still capable of producing a heat shock response, these cells received a heat shock (40°C, 41°C, and 42°C) for one hour and were allowed to recover for six hours at 37°C. During heat shock and recovery periods, lectin exposure was continued.
RESULTS—Constitutive levels of HSPs were measured in the intestinal cells of lactalbumin fed (control) rats, as may be expected from the function of this tissue. However, in PHA and WGA fed rats a marked decline in the binding of antibodies against several HSPs to the intestinal epithelium was found. These results were confirmed by in vitro experiments using differentiated Caco-2 cells exposed to PHA-E4 and WGA. However, after exposure to lectins, these cells were still capable

  18. Plasma mutant α-galactosidase A protein and globotriaosylsphingosine level in Fabry disease.

    PubMed

    Tsukimura, Takahiro; Nakano, Sachie; Togawa, Tadayasu; Tanaka, Toshie; Saito, Seiji; Ohno, Kazuki; Shibasaki, Futoshi; Sakuraba, Hitoshi

    2014-01-01

    Fabry disease is an X-linked genetic disorder characterized by deficient activity of α-galactosidase A (GLA) and accumulation of glycolipids, and various GLA gene mutations lead to a wide range of clinical phenotypes from the classic form to the later-onset one. To investigate the biochemical heterogeneity and elucidate the basis of the disease using available clinical samples, we measured GLA activity, GLA protein and accumulated globotriaosylsphingosine (Lyso-Gb3), a biomarker of this disease, in plasma samples from Fabry patients. The analysis revealed that both the enzyme activity and the protein level were apparently decreased, and the enzyme activity was well correlated with the protein level in many Fabry patients. In these cases, a defect of biosynthesis or excessive degradation of mutant GLAs should be involved in the pathogenesis, and the residual protein level would determine the accumulation of Lyso-Gb3 and the severity of the disease. However, there are some exceptional cases, i.e., ones harboring p.C142Y, p.R112H and p.M296I, who exhibit a considerable amount of GLA protein. Especially, a subset of Fabry patients with p.R112H or p.M296I has been attracted interest because the patients exhibit almost normal plasma Lyso-Gb3 concentration. Structural analysis revealed that C142Y causes a structural change at the entrance of the active site. It will lead to a complete enzyme activity deficiency, resulting in a high level of plasma Lyso-Gb3 and the classic Fabry disease. On the other hand, it is thought that R112H causes a relatively large structural change on the molecular surface, and M296I a small one in a restricted region from the core to the surface, both the structural changes being far from the active site. These changes will cause not only partial degradation but also degeneration of the mutant GLA proteins, and the degenerated enzymes exhibiting small and residual activity remain and probably facilitate degradation of Lyso-Gb3 in plasma, leading

  19. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

    PubMed Central

    Uno, Miyuki; Oba-Shinjo, Sueli Mieko; Camargo, Anamaria Aranha; Moura, Ricardo Pereira; de Aguiar, Paulo Henrique; Cabrera, Hector Navarro; Begnami, Marcos; Rosemberg, Sérgio; Teixeira, Manoel Jacobsen; Marie, Suely Kazue Nagahashi

    2011-01-01

    OBJECTIVES: 1) To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT) promoter to its gene and protein expression levels in glioblastoma and 2) to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry. RESULTS: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001). However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297). The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing), and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis. DISCUSSION AND CONCLUSION: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue. PMID:22012047

  20. Optimal dietary protein level improved growth, disease resistance, intestinal immune and physical barrier function of young grass carp (Ctenopharyngodon idella).

    PubMed

    Xu, Jing; Wu, Pei; Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

    2016-08-01

    This study investigated the effects of dietary proteins on the growth, disease resistance, intestinal immune and physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg(-1) diet) for 8 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila and mortalities were recorded for 14 days. The results indicated that optimal dietary protein levels: increased the production of antibacterial components, up-regulated anti-inflammatory cytokines, inhibitor of κBα, target of rapamycin and ribosomal protein S6 kinases 1 mRNA levels, whereas down-regulated pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) P65, NF-κB P52, c-Rel, IκB kinase β, IκB kinase γ and eIF4E-binding proteins 2 mRNA levels in three intestinal segments of young grass carp (P < 0.05), suggesting that optimal dietary protein level could enhance fish intestinal immune barrier function; up-regulated the mRNA levels of tight junction complexes, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and NF-E2-related factor 2, and increased the activities and mRNA levels of antioxidant enzymes, whereas down-regulated myosin light chain kinase, cysteinyl aspartic acid-protease 2, 3, 7, 8, 9, fatty acid synthetase ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein, p38 mitogen-activated protein kinase, c-Jun N-terminal protein kinase and Kelch-like-ECH-associated protein 1b mRNA levels, and decreased reactive oxygen species, malondialdehyde and protein carbonyl contents in three intestinal segments of young grass carp (P < 0.05), indicating that optimal dietary protein level could improve fish intestinal physical barrier function. Finally, the optimal dietary protein levels for the growth performance (PWG) and against enteritis

  1. Effects of fat and protein levels on foraging preferences of tannin in scatter-hoarding rodents.

    PubMed

    Wang, Bo; Chen, Jin

    2012-01-01

    Both as consumers and dispersers of seeds, scatter-hoarding rodents often play an important role in the reproductive ecology of many plant species. However, the seeds of many plant species contain tannins, which are a diverse group of water-soluble phenolic compounds that have a high affinity for proteins. The amount of tannins in seeds is expected to affect rodent foraging preferences because of their major impact on rodent physiology and survival. However, variable results have been obtained in studies that evaluated the effects of tannin on rodent foraging behavior. Hence, in this study, we aimed to explain these inconsistent results and proposed that a combination of seed traits might be important in rodent foraging behavior, because it is difficult to distinguish between the effects of individual traits on rodent foraging behavior and the interactions among them. By using a novel artificial seed system, we manipulated seed tannin and fat/protein levels to examine directly the univariate effects of each component on the seed preferences of free-ranging forest rats (Apodemus latronum and Apodemus chevrieri) during the behavioral process of scatter hoarding. Our results showed that both tannin and fat/protein had significant effects on rodent foraging behavior. Although only a few interactive effects of tannin and fat/protein were recorded, higher concentrations of both fat and protein could attenuate the exclusion of seeds with higher tannin concentrations by rodents, thus influencing seed fate. Furthermore, aside from the concentrations of tannin, fat, and protein, numerous other traits of plant seeds may also influence rodent foraging behavior. We suggest that by clarifying rodent foraging preferences, a better understanding of the evolution of plant seed traits may be obtained because of their strong potential for selective pressure.

  2. Wolbachia Endosymbionts Modify Drosophila Ovary Protein Levels in a Context-Dependent Manner

    PubMed Central

    Christensen, Steen; Pérez Dulzaides, Ricardo; Hedrick, Victoria E.; Momtaz, A. J. M. Zehadee; Nakayasu, Ernesto S.; Paul, Lake N.

    2016-01-01

    ABSTRACT Endosymbiosis is a unique form of interaction between organisms, with one organism dwelling inside the other. One of the most widespread endosymbionts is Wolbachia pipientis, a maternally transmitted bacterium carried by insects, crustaceans, mites, and filarial nematodes. Although candidate proteins that contribute to maternal transmission have been identified, the molecular basis for maternal Wolbachia transmission remains largely unknown. To investigate transmission-related processes in response to Wolbachia infection, ovarian proteomes were analyzed from Wolbachia-infected Drosophila melanogaster and D. simulans. Endogenous and variant host-strain combinations were investigated. Significant and differentially abundant ovarian proteins were detected, indicating substantial regulatory changes in response to Wolbachia. Variant Wolbachia strains were associated with a broader impact on the ovary proteome than endogenous Wolbachia strains. The D. melanogaster ovarian environment also exhibited a higher level of diversity of proteomic responses to Wolbachia than D. simulans. Overall, many Wolbachia-responsive ovarian proteins detected in this study were consistent with expectations from the experimental literature. This suggests that context-specific changes in protein abundance contribute to Wolbachia manipulation of transmission-related mechanisms in oogenesis. IMPORTANCE Millions of insect species naturally carry bacterial endosymbionts called Wolbachia. Wolbachia bacteria are transmitted by females to their offspring through a robust egg-loading mechanism. The molecular basis for Wolbachia transmission remains poorly understood at this time, however. This proteomic study identified specific fruit fly ovarian proteins as being upregulated or downregulated in response to Wolbachia infection. The majority of these protein responses correlated specifically with the type of host and Wolbachia strain involved. This work corroborates previously identified

  3. Effect of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of certain plasma enzymes in CCl4-induced liver injury in low-protein fed rats.

    PubMed

    Nkosi, C Z; Opoku, A R; Terblanche, S E

    2005-04-01

    The effects of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of lactate dehydrogenase (LD), alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) against carbon tetrachloride (CCl4)-induced acute liver injury in low-protein fed rats were investigated. A group of male Sprague-Dawley rats maintained on a low-protein diet for 5 days were divided into three subgroups. Two subgroups were injected with carbon tetrachloride and the other group with an equivalent amount of olive oil. Two hours after CCl4 intoxication one of the two subgroups was administered with pumpkin seed protein isolate. All three subgroups of rats were maintained on the low-protein diet for the duration of the investigation. Groups of rats from the different subgroups were killed at 24, 48 and 72 h after their respective treatments. After 5 days on the low-protein diet the activity levels of all four enzymes were significantly higher than their counterparts on a normal balanced diet. CCl4 intoxication resulted in significant increases in the activity levels of all four enzymes investigated. The administration of pumpkin seed protein isolate after CCl4 intoxication resulted in significantly reduced activity levels of all four enzymes. It is concluded that pumpkin seed protein isolate administration was effective in alleviating the detrimental effects associated with protein malnutrition.

  4. Intermediate divergence levels maximize the strength of structure-sequence correlations in enzymes and viral proteins.

    PubMed

    Jackson, Eleisha L; Shahmoradi, Amir; Spielman, Stephanie J; Jack, Benjamin R; Wilke, Claus O

    2016-07-01

    Structural properties such as solvent accessibility and contact number predict site-specific sequence variability in many proteins. However, the strength and significance of these structure-sequence relationships vary widely among different proteins, with absolute correlation strengths ranging from 0 to 0.8. In particular, two recent works have made contradictory observations. Yeh et al. (Mol. Biol. Evol. 31:135-139, 2014) found that both relative solvent accessibility (RSA) and weighted contact number (WCN) are good predictors of sitewise evolutionary rate in enzymes, with WCN clearly out-performing RSA. Shahmoradi et al. (J. Mol. Evol. 79:130-142, 2014) considered these same predictors (as well as others) in viral proteins and found much weaker correlations and no clear advantage of WCN over RSA. Because these two studies had substantial methodological differences, however, a direct comparison of their results is not possible. Here, we reanalyze the datasets of the two studies with one uniform analysis pipeline, and we find that many apparent discrepancies between the two analyses can be attributed to the extent of sequence divergence in individual alignments. Specifically, the alignments of the enzyme dataset are much more diverged than those of the virus dataset, and proteins with higher divergence exhibit, on average, stronger structure-sequence correlations. However, the highest structure-sequence correlations are observed at intermediate divergence levels, where both highly conserved and highly variable sites are present in the same alignment.

  5. Lung Surfactant Levels are Regulated by Ig-Hepta/GPR116 by Monitoring Surfactant Protein D

    PubMed Central

    Fukuzawa, Taku; Ishida, Junji; Kato, Akira; Ichinose, Taro; Ariestanti, Donna Maretta; Takahashi, Tomoya; Ito, Kunitoshi; Abe, Jumpei; Suzuki, Tomohiro; Wakana, Shigeharu; Fukamizu, Akiyoshi; Nakamura, Nobuhiro; Hirose, Shigehisa

    2013-01-01

    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. PMID:23922714

  6. A High-throughput Screening Assay for Determining Cellular Levels of Total Tau Protein

    PubMed Central

    Dehdashti, Seameen J.; Zheng, Wei; Gever, Joel R.; Wilhelm, Robert; Nguyen, Dac-Trung; Sittampalam, Gurusingham; McKew, John C.; Austin, Christopher P.; Prusiner, Stanley B.

    2014-01-01

    The microtubule-associated protein (MAP) tau has been implicated in the pathology of numerous neurodegenerative diseases. In the past decade, the hyperphosphorylated and aggregated states of tau protein have been important targets in the drug discovery field for the potential treatment of Alzheimer’s disease. Although several compounds have been reported to reduce the hyperphosphorylated state of tau or impact the stabilization of tau, their therapeutic activities are still to be validated. Recently, reduction of total cellular tau protein has emerged as an alternate intervention point for drug development and a potential treatment of tauopathies. We have developed and optimized a homogenous assay, using the AlphaLISA and HTRF assay technologies, for the quantification of total cellular tau protein levels in the SH-SY5Y neuroblastoma cell line. The signal-to-basal ratios were 375 and 5.3, and the Z’ factors were 0.67 and 0.60 for the AlphaLISA and HTRF tau assays, respectively. The clear advantages of this homogeneous tau assay over conventional total tau assays, such as ELISA and Western blot, are the elimination of plate wash steps and miniaturization of the assay into 1536-well plate format for the ultra–high-throughput screening of large compound libraries. PMID:23905996

  7. Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

    SciTech Connect

    Kanginakudru, Sriramana; DeSmet, Marsha; Thomas, Yanique; Morgan, Iain M.; Androphy, Elliot J.

    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.

  8. Serum levels of bone Gla-protein in inhabitants exposed to environmental cadmium

    SciTech Connect

    Kido, T.; Honda, R.; Tsuritani, I.; Ishizaki, M.; Yamada, Y.; Nakagawa, H.; Nogawa, K.; Dohi, Y. )

    1991-01-01

    Serum levels of bone Gla-protein (BGP)--the vitamin K-dependent CA2(+)-binding protein--were evaluated in 76 cadmium (Cd)-exposed subjects with renal tubular dysfunction (32 men, 44 women) and 133 nonexposed subjects (53 men, 80 women). Serum BGP levels were higher in the Cd-exposed subjects than in nonexposed subjects. Significant correlations between BGP and each index measured by bone microdensitometry (MD), serum alkaline phosphatase activity, and Cd in blood and urine were found. For all of the Cd-exposed and nonexposed men and women, BGP showed a significant standard partial regression coefficient (multiple regression analysis) with the metacarpal index (MCI), which was one of the MD indicators. Bone Gla-protein also correlated significantly with urinary beta 2-microglobulin in the men and with serum creatinine in the women. Serum BGP values strongly reflect the degree of bone damage and also reflect, although less strongly, the degree of renal damage induced by exposure to Cd.

  9. Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

    SciTech Connect

    Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; Razooky, Brandon S.; Simpson, Michael L.; Raj, Arjun; Weinberger, Leor S.

    2016-07-28

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.

  10. Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

    DOE PAGES

    Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; ...

    2016-07-28

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

  11. Retroviral vectors elevate coexpressed protein levels in trans through cap-dependent translation.

    PubMed

    Gou, Yongqiang; Byun, Hyewon; Zook, Adam E; Singh, Gurvani B; Nash, Andrea K; Lozano, Mary M; Dudley, Jaquelin P

    2015-03-17

    Retroviruses cause immunodeficiency and cancer but also are used as vectors for the expression of heterologous genes. Nevertheless, optimal translation of introduced genes often is not achieved. Here we show that transfection into mammalian cells of lentiviral or gammaretroviral vectors, including those with specific shRNAs, increased expression of a cotransfected gene relative to standard plasmid vectors. Levels of most endogenous cellular proteins were unchanged. Transfer of lentiviral vector sequences into a standard plasmid conferred the ability to give increased expression of cotransfected genes (superinduction). Superinduction by the retroviral vector was not dependent on the cell type or species, the type of reporter gene, or the method of transfection. No differences were detected in the IFN, unfolded protein, or stress responses in the presence of retroviral vectors. RT-PCRs revealed that RNA levels of cotransfected genes were unchanged during superinduction, yet Western blotting, pulse labeling, and the use of bicistronic vectors showed increased cap-dependent translation of cointroduced genes. Expression of the mammalian target of rapamycin (mTOR) kinase target 4E-BP1, but not the mTOR inhibitor Torin 1, preferentially inhibited superinduction relative to basal protein expression. Furthermore, transcription of lentiviral vector sequences from a doxycycline-inducible promoter eliminated superinduction, consistent with a DNA-triggered event. Thus, retroviral DNA increased translation of cointroduced genes in trans by an mTOR-independent signaling mechanism. Our experiments have broad applications for the design of retroviral vectors for transfections, DNA vaccines, and gene therapy.

  12. C-reactive protein and alpha 1-acid glycoprotein levels in dogs infected with Ehrlichia canis.

    PubMed Central

    Rikihisa, Y; Yamamoto, S; Kwak, I; Iqbal, Z; Kociba, G; Mott, J; Chichanasiriwithaya, W

    1994-01-01

    To elucidate whether acute-phase protein responses occur in dogs infected with Ehrlichia canis, C-reactive protein (CRP) and alpha 1-acid glycoprotein (AAG) levels were serially measured in the plasma of five dogs experimentally inoculated with E. canis and 10 sham-inoculated or noninoculated control dogs. The CRP concentration was measured by a canine-specific capture enzyme-linked immunosorbent assay, and the AAG concentration was measured by a canine-specific radial immunodiffusion method. In all E. canis-inoculated dogs, a 3.3- to 6.5-fold increase in the plasma CRP concentration and a 1.9- to 8.6-fold increase in the plasma AAG concentration over the preinoculation level occurred at days 4 to 6 postexposure. Despite the persistence of E. canis and high antibody titers, both CRP and AAG concentrations gradually declined to preexposure levels by day 34 postexposure. E. canis-infected dogs had mild and transient clinical signs which resolved without treatment by day 14 postexposure. The CRP and AAG concentrations in control inoculated or nontreated dogs remained within the normal range throughout the experimental period. Of 12 dogs naturally infected with E. canis, 75% had greater than 50 micrograms of CRP per ml and 83% had greater than 500 micrograms of AAG per ml. All of these 12 dogs had chronic and severe clinical signs of canine ehrlichiosis. Thus, elevations in the levels of acute-phase proteins occur in both acute and chronic canine ehrlichiosis. Determination of CRP and AAG concentrations may help in assessing the severity of inflammatory damage in dogs with E. canis infections. PMID:8027343

  13. Nitrous oxide plus isoflurane induces apoptosis and increases β-amyloid protein levels

    PubMed Central

    Zhen, Yu; Dong, Yuanlin; Wu, Xu; Xu, Zhipeng; Lu, Yan; Zhang, Yiying; Norton, David; Tian, Ming; Li, Shuren; Xie, Zhongcong

    2009-01-01

    Background Some anesthetics have been suggested to induce neurotoxicity including promotion of Alzheimer’s disease neuropathogenesis. Nitrous oxide and isoflurane are common anesthetics. Here, we set out to assess effects of nitrous oxide and/or isoflurane on apoptosis and β-amyloid (Aβ) levels in H4 human neuroglioma cells and primary neurons from naïve mice. Methods The cells or neurons were exposed to 70% nitrous oxide and/or 1% isoflurane for six hours. The cells or neurons and conditioned media were harvested at the end of the treatment. Caspase-3 activation, apoptosis, processing of amyloid precursor protein, and Aβ levels were determined. Results Treatment with a combination of 70% nitrous oxide and 1% isoflurane for six hours induced caspase-3 activation and apoptosis in H4 naïve cells and primary neurons from naïve mice. The 70% nitrous oxide plus 1% isoflurane, but neither alone, for six hours induced caspase-3 activation and apoptosis, and increased levels of β-site amyloid precursor protein-cleaving enzyme and Aβ in H4-amyloid precursor protein cells. In addition, the nitrous oxide plus isoflurane-induced Aβ generation was reduced by a broad caspase inhibitor Z-VAD. Finally, the nitrous oxide plus isoflurane-induced caspase-3 activation was attenuated by γ-secretase inhibitor L-685,458, but potentiated by exogenously added Aβ. Conclusion These results suggest that common anesthetics nitrous oxide plus isoflurane may promote neurotoxicity by inducing apoptosis and increasing Aβ levels. The generated Aβ may further potentiate apoptosis to form another round of apoptosis and Aβ generation. More studies, especially the in vivo confirmation of these in vitro findings, are needed. PMID:19741497

  14. Mutations in cystathionine beta-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans.

    PubMed

    Jakubowski, Hieronim; Boers, Godfried H J; Strauss, Kevin A

    2008-12-01

    Severely elevated plasma homocysteine (Hcy) levels observed in genetic disorders of Hcy metabolism are associated with pathologies in multiple organs and lead to premature death due to vascular complications. In addition to elevating plasma Hcy, mutations in cystathionine beta-synthase (CBS) or methylenetetrahydrofolate reductase (MTHFR) gene lead to markedly elevated levels of circulating Hcy-thiolactone. The thiooester chemistry of Hcy-thiolactone underlies its ability to form isopeptide bonds with protein lysine residues (N-Hcy-protein), which may impair or alter the protein's function. However, it was not known whether genetic deficiencies in Hcy metabolism affect N-Hcy-protein levels in humans. Here we show that plasma N-Hcy-protein levels are significantly elevated in CBS- and MTHFR-deficient patients. We also show that CBS-deficient patients have significantly elevated plasma levels of prothrombotic N-Hcy-fibrinogen. These results provide a possible explanation for increased atherothrombosis observed in CBS-deficient patients.

  15. COPD assessment test score and serum C-reactive protein levels in stable COPD patients

    PubMed Central

    Kang, Hyung Koo; Kim, Kang; Lee, Hyun; Jeong, Byeong-Ho; Koh, Won-Jung; Park, Hye Yun

    2016-01-01

    Background An eight-item questionnaire of the COPD assessment test (CAT) is widely used to quantify the impact of COPD on the patient’s health status. C-reactive protein (CRP) is associated with disease severity and adverse health outcomes of patients with COPD. This study aimed to evaluate the relationship between CAT score and serum CRP levels in stable COPD patients. Methods We evaluated the medical records of 226 patients with CAT and serum CRP measured within a week at Samsung Medical Center between October 2013 and October 2015. Results Serum CRP levels had a significantly positive relationship with CAT score (Spearman’s r=0.20, P=0.003). Patients with elevated serum CRP levels (>0.3 mg/dL) were significantly more likely to have CAT scores of ≥14. The adjusted odds ratio for elevated serum CRP levels in total CAT score was 1.06 (95% confidence interval, 1.02–1.09). Among CAT components, cough (adjusted P=0.005), phlegm (adjusted P=0.001), breathlessness going up hills/stairs (adjusted P=0.005), low confidence leaving home (adjusted P=0.002), and feeling low in energy (adjusted P=0.019) were independently associated with elevated serum CRP levels. Conclusion In stable COPD patients, serum CRP levels were independently associated with total CAT score and CAT components related to respiratory symptoms, confidence leaving home, and energy. PMID:27994452

  16. Efficacy of erythrocyte sedimentation rate and C-reactive protein level in determining periprosthetic hip infections.

    PubMed

    Costa, Christopher R; Johnson, Aaron J; Naziri, Qais; Maralunda, German A; Delanois, Ronald E; Mont, Michael A

    2012-04-01

    The diagnosis of periprosthetic hip infections is often challenging. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) level blood laboratory tests are commonly used to aid in the diagnosis. We studied the sensitivity, specificity, and false-negative rates of ESR and CRP level in a prospective group of patients who underwent revision total hip arthroplasty between 2000 and 2008. Seventy-seven patients with periprosthetic hip infections and ESR and CRP data were identified. Chi-square analysis was performed to determine the significance of false-negatives, compared with sex, body mass index, primary diagnosis, infection type, and immunity status. ESR had 89% sensitivity and 69% specificity. CRP level had 93% sensitivity and 40% specificity. The false-negative rate was 10.8% for ESR and 7% for CRP level. The false-negative rate for ESR and CRP level combined (with either result positive) was 3%. All false-negatives in the combined group were immunocompromised. Chi-square analysis did not find a significant correlation between false-negatives and any other variables. ESR and CRP level are useful in the diagnosis of periprosthetic hip infections. Ordering these tests concurrently reduces the chance of false-negative results.

  17. Mechanical spectroscopy of retina explants at the protein level employing nanostructured scaffolds.

    PubMed

    Mayazur Rahman, S; Reichenbach, Andreas; Zink, Mareike; Mayr, Stefan G

    2016-04-14

    Development of neuronal tissue, such as folding of the brain, and formation of the fovea centralis in the human retina are intimately connected with the mechanical properties of the underlying cells and the extracellular matrix. In particular for neuronal tissue as complex as the vertebrate retina, mechanical properties are still a matter of debate due to their relation to numerous diseases as well as surgery, where the tension of the retina can result in tissue detachment during cutting. However, measuring the elasticity of adult retina wholemounts is difficult and until now only the mechanical properties at the surface have been characterized with micrometer resolution. Many processes, however, such as pathological changes prone to cause tissue rupture and detachment, respectively, are reflected in variations of retina elasticity at smaller length scales at the protein level. In the present work we demonstrate that freely oscillating cantilevers composed of nanostructured TiO2 scaffolds can be employed to study the frequency-dependent mechanical response of adult mammalian retina explants at the nanoscale. Constituting highly versatile scaffolds with strong tissue attachment for long-term organotypic culture atop, these scaffolds perform damped vibrations as fingerprints of the mechanical tissue properties that are derived using finite element calculations. Since the tissue adheres to the nanostructures via constitutive proteins on the photoreceptor side of the retina, the latter are stretched and compressed during vibration of the underlying scaffold. Probing mechanical response of individual proteins within the tissue, the proposed mechanical spectroscopy approach opens the way for studying tissue mechanics, diseases and the effect of drugs at the protein level.

  18. Effects of dietary protein level on growth performance and nitrogen excretion of dairy heifers

    PubMed Central

    Zhang, Bin; Wang, Chong; Liu, He; Liu, Jianxin; Liu, Hongyun

    2017-01-01

    Objective Protein supplementation is costly and can result in excess nitrogen (N) excretion. The objective of this study was to evaluate the effects of feeding different levels of dietary protein on average daily gain, body size, rumen fermentation, and nitrogen excretion of 8 to 10 month-old Holstein heifers. Methods Thirty-six Holstein heifers were divided into 12 blocks according to age (273±6.2 d) and were randomly assigned to diets containing a low (10.2% dry matter [DM]), medium (11.9% DM), or high (13.5% DM) level of dietary crude protein (CP). All diets contained approximately 70% roughage and 30% concentrate with similar dietary metabolizable energy (ME) content (2.47 Mcal/kg). Results Dry matter intake did not differ among the treatments, and average daily gain increased with the increasing dietary protein, 0.79, 0.95, 0.97 kg/d for low, medium, and high group, respectively. Body height increased linearly with increasing dietary CP but no other significant differences in body dimensions were found among the treatments. The increased ratios of dietary CP improved the rate of rear teat length growth remarkably (p<0.05). There was no difference in rumen pH or ruminal major volatile fatty acid (acetate, propionate, and butyrate) concentration among the 3 diets, but rumen ammonia-N concentration increased with the higher dietary CP (p<0.05). Increasing N intake led to increased total N excretion; urinary N excretion was significantly increased (p<0.05) but fecal N excretion was similar among the treatments. Conclusion These data suggest that the diet containing 11.9% CP (ME 2.47 Mcal/kg) could meet the maintenance and growth requirements of 9 to 11 month-old Holstein heifers gaining approximately 0.9 kg/d. PMID:27554361

  19. EFFECT OF DIETARY PROTEIN AND CARBOHYDRATE LEVELS ON WEIGHT GAIN AND GONAD PRODUCTION IN THE SEA URCHIN LYTECHINUS VARIEGATUS.

    PubMed

    Heflin, Laura E; Gibbs, Victoria K; Powell, Mickie L; Makowsky, Robert; Lawrence, John M; Lawrence, Addison L; Watts, Stephen A

    2012-08-15

    Adult Lytechinus variegatus were fed eight formulated diets with different protein (ranging from 12 to 36%) and carbohydrate (ranging from 21 to 39 %) levels. Each sea urchin (n = 8 per treatment) was fed a daily sub-satiation ration of 1.5% of average body weight for 9 weeks. Akaike information criterion analysis was used to compare six different hypothesized dietary composition models across eight growth measurements. Dietary protein level and protein: energy ratio were the best models for prediction of total weight gain. Diets with the highest (> 68.6 mg P kcal(--1)) protein: energy ratios produced the most wet weight gain after 9 weeks. Dietary carbohydrate level was a poor predictor for most growth parameters examined in this study. However, the model containing a protein × carbohydrate interaction effect was the best model for protein efficiency ratio (PER). PER decreased with increasing dietary protein level, more so at higher carbohydrate levels. Food conversion ratio (FCR) was best modeled by total dietary energy levels: Higher energy diets produced lower FCRs. Dietary protein level was the best model of gonad wet weight gain. These data suggest that variations in dietary nutrients and energy differentially affect organismal growth and growth of body components.

  20. Elevation of C-reactive protein levels in patients with transfusion-related acute lung injury

    PubMed Central

    Kapur, Rick; Kim, Michael; Rondina, Matthew T.; Porcelijn, Leendert; Semple, John W.

    2016-01-01

    Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities and is characterized by the onset of acute respiratory distress within six hours following blood transfusion. In most cases, donor antibodies are suggested to be involved, however, the pathogenesis is poorly understood. A two-hit model is generally assumed to underlie TRALI pathogenesis where the first hit consists of a patient predisposing factor such as inflammation and the second hit is due to donor antibodies present in the transfused blood. We recently demonstrated that the acute phase protein C-reactive protein (CRP) could enhance murine anti-major histocompatibility complex (MHC) class I-mediated TRALI. Whether CRP is increased in human TRALI patients which would support its role as a risk factor for human TRALI, is currently unknown. For that purpose, we measured CRP levels in the plasma of human TRALI patients and found CRP levels to be significantly elevated compared to transfused control patients. These data support the notion that CRP may be a novel first hit risk factor in human TRALI and that modulation of CRP levels could be an effective therapeutic strategy for this serious adverse event of transfusion. PMID:27793007

  1. Cold induced changes in lipid, protein and carbohydrate levels in the tropical insect Gromphadorhina coquereliana.

    PubMed

    Chowanski, Szymon; Lubawy, Jan; Spochacz, Marta; Ewelina, Paluch; Grzegorz, Smykalla; Rosinski, Grzegorz; Slocinska, Malgorzata

    2015-05-01

    Insects cope with thermal stressors using mechanisms such as rapid cold hardening and acclimation. These mechanisms have been studied in temperate insects, but little is known about their use by tropical insects in response to cold stress. Here, we investigated whether cold stress (1×8 h and 3×8 h at 4°C) triggers a metabolic response in the Madagascar cockroach Gromphadorhina coquereliana. We examined the effects of cold on the levels of selected metabolites in the fat body tissue of G. coquereliana. After cold exposure, we found that the quantity of total protein increased significantly in the insect fat body, whereas glycogen decreased slightly. Using antibodies, we observed upregulation of AQP-like proteins and changes in the HSP70 levels in the fat body of G. coquereliana when exposed to cold. We also examined the content and nature of the free sugars in the G. coquereliana hemolymph and discovered an increase in the levels of polyols and glucose in response to cold stress. These results suggest an important role of the fat body tissue of tropical insects upon cold exposure.

  2. A Screen for Regulators of Survival of Motor Neuron Protein Levels

    PubMed Central

    Makhortova, Nina R.; Hayhurst, Monica; Cerqueira, Antonio; Sinor-Anderson, Amy D.; Zhao, Wen-Ning; Heiser, Patrick W.; Arvanites, Anthony C.; Davidow, Lance S.; Waldon, Zachary O.; Steen, Judith A.; Lam, Kelvin; Ngo, Hien D.; Rubin, Lee L.

    2011-01-01

    The motor neuron disease Spinal Muscular Atrophy (SMA) results from mutations that lead to low levels of the ubiquitously expressed protein Survival of Motor Neuron (SMN). Ever-increasing data suggest that therapeutics that elevate SMN may be effective in treating SMA. We executed an image-based screen of annotated chemical libraries and discovered multiple classes of compounds that were able to increase cellular SMN. Among the most important was the RTK/PI3K/AKT/GSK-3 signaling cascade. Chemical inhibitors of GSK-3, as well as shRNAs directed against this target, elevate SMN levels primarily by stabilizing the protein. Of particular significance is that GSK-3 chemical inhibitors were also effective in motor neurons, not only in elevating SMN levels, but also in blocking the death that was produced when SMN was acutely reduced by a SMN-specific shRNA. Thus, we have established a screen capable of detecting drug-like compounds that correct the main phenotypic change that underlies SMA. PMID:21685895

  3. The evaluation of increase in hemodialysis frequency on C-reactive protein levels and nutritional status.

    PubMed

    Rashidi, Ali Akbar; Soleimani, Ali Reza; Nikoueinejad, Hassan; Sarbolouki, Shokooh

    2013-03-16

    Malnutrition and inflammation are the most important causes of cardiovascular disease in hemodialysis patients. This study was conducted to evaluate the effect of increase in hemodialysis frequency on C-reactive protein (CRP) level and nutritional markers in contrast to previous routine method. 18 hemodialysis patients with a mean age of 53±16 years were randomly selected in this before-and-after clinical trial. The patients under a standard hemodialysis of 3 times/4 h per week were converted to 4 times/4 h for a period of 6 weeks. The CRP, albumin, triglyceride, total cholesterol, LDL, HDL serum levels, anthropometric indices and 24-h diet recall intake was assessed before and after of the period. The data were analyzed using paired t-test, and P-value less than 0.05 was considered significant. All patients completed the study. Mean weight, body mass index and serum albumin increased while serum CRP level decreased significantly after the intervention (P<0.03). Triglyceride, total cholesterol, LDL, HDL, as well as energy, protein and fat intake had no significant change before and after the study. Increase in dialysis frequency decreased systemic inflammation and improved the nutritional state of hemodialysis patients. Therefore, it may decrease the risk of cardiovascular events in these patients.

  4. Evaluation of behaviour in stabled draught horse foals fed diets with two protein levels.

    PubMed

    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 <2%. Concerning diet, foals fed high protein spent more time in movement (19.62±0.73% of day v. 10.45±0.73% in low-protein (LP) foals; P⩽0.001), whereas the LP group increased resting (43.42±1.12% v. 38.02±1.12%; P⩽0.001). No stereotypies were found, and daily activity followed the typical values for 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.

  5. Mechanical Folding and Unfolding of Protein Barnase at the Single-Molecule Level

    PubMed Central

    Alemany, Anna; Rey-Serra, Blanca; Frutos, Silvia; Cecconi, Ciro; Ritort, Felix

    2016-01-01

    The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale. PMID:26745410

  6. Resveratrol Administration Increases Transthyretin Protein Levels, Ameliorating AD Features: The Importance of Transthyretin Tetrameric Stability

    PubMed Central

    Santos, Luís Miguel; Rodrigues, Daniela; Alemi, Mobina; Silva, Sara Costa; Ribeiro, Carlos Alexandre; Cardoso, Isabel

    2016-01-01

    Previous in vivo work showed that resveratrol has beneficial effects in Alzheimer’s disease (AD) pathology, resulting in increased expression of transthyretin (TTR). TTR binds amyloid-beta (Aβ) peptide, avoiding its aggregation and toxicity, and is reduced in the cerebrospinal fluid (CSF) and plasma in AD. Further, resveratrol binds TTR, stabilizing the native TTR tetrameric structure. To further explore the mechanism of neuroprotection conferred by TTR in AD, resveratrol was administered in the diet to 5- to 8-month-old AD transgenic female mice carrying just 1 copy of the mouse TTR gene for 2 months. Effects in brain Aβ burden were evaluated by immunohistochemistry, and total brain Aβ levels by ELISA, showing a striking decrease in both parameters in treated animals. In addition, total brain lipoprotein-related receptor protein 1 (LRP1) levels were increased in treated animals, although its gene expression was unaltered. To further understand the mechanism(s) underlying such improvement in AD features, we measured TTR plasma levels, showing that TTR increased in resveratrol-treated mice, whereas liver TTR gene transcription was not altered. These results strengthen the stability hypothesis, which postulates that TTR is unstable in AD, leading to accelerated clearance and lower levels. Therefore, resveratrol, which stabilizes the TTR tetramer results in TTR-normalized clearance, increases the protein plasma levels. In turn, stabilized TTR binds more strongly to Aβ peptide, avoiding its aggregation. Our results represent a step forward in the understanding of the mechanism underlying TTR protection in AD and highlight the possibility of using TTR stabilization as a therapeutic target in AD. PMID:27385446

  7. The effect of elevated plasma phenylalanine levels on protein synthesis rates in adult rat brain.

    PubMed Central

    Dunlop, D S; Yang, X R; Lajtha, A

    1994-01-01

    Increasing the plasma phenylalanine concentration to levels as high as 0.560-0.870 mM (over ten times normal levels) had no detectable effect on the rate of brain protein synthesis in adult rats. The average rates for 7-week-old rats were: valine, 0.58 +/- 0.05%/h, phenylalanine, 0.59 +/- 0.06%/h, and tyrosine, 0.60 +/- 0.09%/h, or 0.59 +/- 0.06%/h overall. Synthesis rates calculated on the basis of the specific activity of the tRNA-bound amino acid were slightly lower (4% lower for phenylalanine) than those based on the brain free amino acid pool. Similarly, the specific activities of valine and phenylalanine in microdialysis fluid from striatum were practically the same as those in the brain free amino acid pool. Thus the specific activities of the valine and phenylalanine brain free pools are good measures of the precursor specific activity for protein synthesis. In any event, synthesis rates, whether based on the specific activities of the amino acids in the brain free pool or those bound to tRNA, were unaffected by elevated levels of plasma phenylalanine. Brain protein synthesis rates measured after the administration of quite large doses of phenylalanine (> 1.5 mumol/g) or valine (15 mumol/g) were in agreement (0.62 +/- 0.01 and 0.65 +/- 0.01%/h respectively) with the rates determined with infusions of trace amounts of amino acids. Thus the technique of stabilizing precursor-specific activity, and pushing values in the brain close to those of the plasma, by the administration of large quantities of precursor, appears to be valid. PMID:8093014

  8. Analysis of porcine peripheral blood mononuclear cells proteome by 2-DE and MS: analytical and biological variability in the protein expression level and protein identification.

    PubMed

    Ramirez-Boo, María; Garrido, Juan J; Ogueta, Samuel; Calvete, Juan J; Gómez-Díaz, Consuelo; Moreno, Angela

    2006-04-01

    In this paper, we present the protein map corresponding to the porcine peripheral blood mononuclear cells (PBMC) to better understand the role of these cells in the pig immune system. To conform the map, the proteins were separated by 2-DE using a 5-8 range pH gradient in IEF and approximately 800 spots were detected. Due to the high level of indeterminate variability associates to the 2-DE, analytical and biological variances were analyzed. The analytical variance was calculated for 50 proteins in three replicate 2-DE gels from the same protein extract whereas the biological variance was determined by comparison of the patterns obtained for the same 50 proteins in different animals. Values of 15.13 and 33.70% were determined for analytical and biological variances, respectively. These average variances will provide a quantified and statistical basis for future proteomic studies directed to evaluate relevant quantitative changes in the biological response. A representative set of the major proteins was subjected to MALDI-TOF analysis and over 75% of the proteins were identified on the basis of their similarity with its human homologue proteins. A large number of cytoskeletal and metabolic proteins were found as well as some proteins related to cell mobility and immunological functions. Finally, other proteins implicated in the cell signaling process, transport or apoptosis were also identified giving a wide overview of the porcine PBMC protein map.

  9. Plasma Levels of Soluble Interleukin 1 Receptor Accessory Protein Are Reduced in Obesity

    PubMed Central

    Attard, Chantal; Kulkarni, Hemant; Cummings, Nik; Diego, Vincent P.; Carless, Melanie A.; Shields, Katherine A.; Johnson, Matthew P.; Kowlessur, Sudhir; Dyer, Thomas D.; Comuzzie, Anthony G.; Almasy, Laura; Zimmet, Paul; Moses, Eric K.; Göring, Harald H. H.; Curran, Joanne E.; Blangero, John; Jowett, Jeremy B. M.

    2014-01-01

    Context: Adipokines actuate chronic, low-grade inflammation through a complex network of immune markers, but the current understanding of these networks is incomplete. The soluble isoform of the IL-1 receptor accessory protein (sIL1RAP) occupies an important position in the inflammatory pathways involved in obesity. The pathogenetic and clinical influences of sIL1RAP are unknown. Objective: The objective of the study was to elucidate whether plasma levels of sIL1RAP are reduced in obesity, using affluent clinical, biochemical, and genetic data from two diverse cohorts. Design, Setting, and Participants: The study was conducted in two cohorts: the San Antonio Family Heart Study (n = 1397 individuals from 42 families) and South Asians living in Mauritius, n = 230). Main Outcome Measures: Plasma sIL1RAP levels were measured using an ELISA. The genetic basis of sIL1RAP levels were investigated using both a large-scale gene expression profiling study and a genome-wide association study. Results: A significant decrease in plasma sIL1RAP levels were observed in obese subjects, even after adjustment for age and sex. The sIL1RAP levels demonstrated a strong inverse association with obesity measures in both populations. All associations were more significant in females. Plasma sIL1RAP levels were significantly heritable, correlated with IL1RAP transcript levels (NM_134470), showed evidence for shared genetic influences with obesity measures and were significantly associated with the rs2885373 single-nucleotide polymorphism (P = 6.7 × 10−23) within the IL1RAP gene. Conclusions: Plasma sIL1RAP levels are reduced in obesity and can potentially act as biomarkers of obesity. Mechanistic studies are required to understand the exact contribution of sIL1RAP to the pathogenesis of obesity. PMID:24915116

  10. Effects of High Estrogen Levels on Monocyte Chemoattractant Protein-1 and Wound Healing

    PubMed Central

    Plackett, Timothy P.; Gregory, Meredith S.; Kovacs, Elizabeth J.

    2015-01-01

    Objective: Herein, we tested the effects of high levels of supplemental estrogen treatment on cutaneous wound healing. Approach: Female mice were implanted with a 17β-estradiol (E2) secreting pellet or placebo before receiving a full-thickness dermal excisional wound. Mice receiving the E2 pellet attained hormone levels that are comparable to those achieved during pregnancy. At 1, 3, and 5 days after injury, the dermal excision wound was examined for their histologic appearance, rate of closure, and chemokine levels. Results: Wound closure, assessed by percent reepithelialization, was slower in E2-treated mice relative to placebo (42.6%±6.6% vs. 70.0%±5.3%, respectively, 3 days after injury). In addition, there was a marked reduction in the subepithelial inflammatory infiltrate and granulation tissue in E2-treated mice relative to placebo. Wound levels of monocyte chemoattractant protein-1 (MCP-1) were increased by 3 days after injury and continued to rise at 5 days after injury in placebo-treated mice (p<0.01). By contrast, MCP-1 levels were significantly reduced at 3 and 5 days after injury in E2-treated mice relative to placebo-treated controls (p<0.01). This attenuation could be reversed by treatment with an estrogen receptor antagonist. Innovation: High levels of estrogen are able to suppress normal wound closure. Conclusion: Dermal wound healing can be altered by manipulating the gonadal steroid hormone levels. In particular, high levels of estrogen can be utilized to slow down the rate of wound healing through a reduction in the inflammatory response. PMID:25713751

  11. In vitro effect of dietary protein level and nondigestible oligosaccharides on feline fecal microbiota.

    PubMed

    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 (P<0.05) by GOS (-9%) after 6 h and by GA (-14%), LAC (-12%), and PEC (-10%) after 24 h. After 24 h, all treatments had resulted in a lower pH versus the CTRL. Putrescine concentrations at 24 h were greater (P<0.05) in cultures treated with FOS (+90%), GOS (+96%), and LAC (+87%). Compared with the CTRL, total VFA were higher (P<0.05) in bottles containing CF (+41%), whereas the acetic to propionic acid ratio was reduced by LAC (-51%; P<0.05). After 24 h, Enterobacteriaceae had been reduced (P<0.05) by LAC and PEC. In a second study, LAC and FOS were selected to be tested in the presence of 2 diets differing in their protein content. There were 6 treatments: low-protein (LP) CTRL with no addition of prebiotics (CTRL-LP), high-protein (HP) CTRL with no addition of prebiotics (CTRL-HP), LP diet plus FOS, CTRL-HP plus FOS, LP diet plus LAC, and CTRL-HP plus LAC. Both FOS and LAC were added to feline fecal cultures at 2 g/L for 24 h incubation. Ammonia at 24 h was affected (P<0.05) by the protein level (36.2 vs. 50.2 mmol/L for LP and HP, respectively). The CTRL-HPs resulted in a higher pH and increased concentrations of biogenic amines were found after 6 and 24 h of incubation (P<0.05); putrescine at 24 h showed an increase (P<0.05) in cultures treated with FOS. Total VFA were influenced (P<0.05) by the protein level (40.9 vs. 32.6 mmol/L for LP and HP, respectively). At 24 h, the CTRL

  12. Effects of Two Different Levels of Dietary Protein on Body Composition and Protein Nutritional Status of Growing Rats

    PubMed Central

    Tirapegui, Julio; Ribeiro, Sandra Maria Lima; Pires, Ivanir Santana de Oliveira; Rogero, Marcelo Macedo

    2012-01-01

    This study aimed to investigate the effect of a high-protein diet on growth, body composition, and protein nutritional status of young rats. Newly-weaned Wistar rats, weighing 45–50 g, were distributed in two experimental groups, according to their diets, which contained 12% (G12) or 26% protein (G26), over a period of 3 weeks. The animals were euthanized at the end of this period and the following analyses were performed: chemical composition of the carcass, proteoglycan synthesis, IGF-I concentration (serum, muscle and cartilage), total tissue RNA, protein concentration (muscle and cartilage) and protein synthesis (muscle and cartilage). The high-protein diet was found to result in a higher fat-free mass and lower fat mass in the carcass, with no difference in growth or protein nutritional status. PMID:23112920

  13. Is cold the new hot? Elevated ubiquitin-conjugated protein levels in tissues of Antarctic fish as evidence for cold-denaturation of proteins in vivo.

    PubMed

    Todgham, Anne E; Hoaglund, Elizabeth A; Hofmann, Gretchen E

    2007-11-01

    Levels of ubiquitin (Ub)-conjugated proteins, as an index of misfolded or damaged proteins, were measured in notothenioid fishes, with both Antarctic (Trematomus bernacchii, T. pennellii, Pagothenia borchgrevinki) and non-Antarctic (Notothenia angustata, Bovichtus variegatus) distributions, as well as non-notothenioid fish from the Antarctic (Lycodichthys dearborni, Family Zoarcidae) and New Zealand (Bellapiscis medius, Family Tripterygiidae), in an effort to better understand the effect that inhabiting a sub-zero environment has on maintaining the integrity of the cellular protein pool. Overall, levels of Ub-conjugated proteins in cold-adapted Antarctic fishes were significantly higher than New Zealand fishes in gill, liver, heart and spleen tissues suggesting that life at sub-zero temperatures impacts protein homeostasis. The highest tissue levels of ubiquitinated proteins were found in the spleen of all fish. Ub conjugate levels in the New Zealand N. angustata, more closely resembled levels measured in other Antarctic fishes than levels measured in other New Zealand species, likely reflecting their recent shared ancestry with Antarctic notothenioids.

  14. Changes in endogenous gene transcript and protein levels in maize plants expressing the soybean ferritin transgene

    PubMed Central

    Kanobe, Milly N.; Rodermel, Steven R.; Bailey, Theodore; Scott, M. Paul

    2013-01-01

    Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, we tested effects of soybean ferritin transgene (SoyFer1, M64337) on transcript and protein levels of endogenous genes in maize. Results showed that the transgene was successfully introduced and expressed in the maize seed endosperm. mRNA abundance of seven tested iron homeostasis genes and seed storage protein genes differed significantly between seed samples positive and negative for the transgene. The PCR negative samples had higher zein and total protein content compared to the positive samples. However, PCR positive samples had significantly higher concentrations of calcium, magnesium, and iron. We have shown that the soybean ferritin transgene affected the expression of native iron homeostasis genes in the maize plant. These results underscore the importance of taking a holistic approach to the evaluation of transgenic events in target plants, comparing the transgenic plant to the untransformed controls. PMID:23785377

  15. Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

    SciTech Connect

    Wilkins, Michael J.; Wrighton, Kelly C.; Nicora, Carrie D.; Williams, Kenneth H.; McCue, Lee Ann; Handley, Kim M.; Miller, C. S.; Giloteaux, L.; Montgomery, A. P.; Lovley, Derek R.; Banfield, Jillian F.; Long, Philip E.; Lipton, Mary S.

    2013-03-05

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.

  16. Dependence of intestinal amino acid uptake on dietary protein or amino acid levels

    SciTech Connect

    Karasov, W.H.; Solberg, D.H.; Diamond, J.M.

    1987-05-01

    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.

  17. Changes in endogenous gene transcript and protein levels in maize plants expressing the soybean ferritin transgene.

    PubMed

    Kanobe, Milly N; Rodermel, Steven R; Bailey, Theodore; Scott, M Paul

    2013-01-01

    Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, we tested effects of soybean ferritin transgene (SoyFer1, M64337) on transcript and protein levels of endogenous genes in maize. Results showed that the transgene was successfully introduced and expressed in the maize seed endosperm. mRNA abundance of seven tested iron homeostasis genes and seed storage protein genes differed significantly between seed samples positive and negative for the transgene. The PCR negative samples had higher zein and total protein content compared to the positive samples. However, PCR positive samples had significantly higher concentrations of calcium, magnesium, and iron. We have shown that the soybean ferritin transgene affected the expression of native iron homeostasis genes in the maize plant. These results underscore the importance of taking a holistic approach to the evaluation of transgenic events in target plants, comparing the transgenic plant to the untransformed controls.

  18. Protein- and tryptophan-restricted diets induce changes in rat gonadal hormone levels.

    PubMed

    Del Angel-Meza, A R.; Feria-Velasco, A; Ontiveros-Martínez, L; Gallardo, L; Gonzalez-Burgos, I; Beas-Zárate, C

    2001-04-01

    The release of gonadotrophic hormones starts at puberty and, along with the subsequent estral cyclicity, is subject to hormonal feedback systems and to the action of diverse neuroactive substances such as gamma amino butyric acid and catecholamines. This study shows the effect of the administration during 40 days of protein-restricted and corn-based (tryptophan- and lysine-deficient) diets on the serotonin concentration in medial hypothalamic fragments as well as in follicle-stimulating luteinizing hormones, 17-beta-estradiol and progesterone serum levels, and estral cyclicity in 60- and 100-day-old rats (young, mature, and in gestation). In young rats, a delay in vaginal aperture development, and a lengthening of the estral cycle to a continuous anestral state was observed, mainly in the group fed corn. This group showed a 25% decrease in the serotonin concentration compared with the protein-restricted group, which exhibited an increase of 9% over the control group. Luteinizing hormone levels decreased in 16% and 13%, whereas follicle-stimulating hormone increased in 13% and 5% in the young animals of restricted groups, respectively, compared with the control group. Serum progesterone levels decreased only in young restricted versus control animals, and no differences were seen among adult and gestational rats. Serum levels of 17-beta-estradiol in restricted animals showed different concentration patterns, mainly in the corn group, which was higher at the 20th gestational day, falling drastically postpartum. The results obtained in this study show serotonin to be a very important factor in the release of gonadotrophic hormones and the start of puberty.

  19. Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors

    PubMed Central

    González, Natalia; Moresco, James J.; Bustos, Francisco; Yates, John R.; Olguín, Hugo C.

    2016-01-01

    Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (re)acquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate. PMID:27144531

  20. Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels

    PubMed Central

    Jiang, Xian-cheng; Bruce, Can; Mar, Jefferson; Lin, Min; Ji, Yong; Francone, Omar L.; Tall, Alan R.

    1999-01-01

    It has been proposed that the plasma phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids and cholesterol from triglyceride-rich lipoproteins (TRL) into high-density lipoproteins (HDL). To evaluate the in vivo role of PLTP in lipoprotein metabolism, we used homologous recombination in embryonic stem cells and produced mice with no PLTP gene expression. Analysis of plasma of F2 homozygous PLTP–/– mice showed complete loss of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, and partial loss of free cholesterol transfer activities. Moreover, the in vivo transfer of [3H]phosphatidylcholine ether from very-low-density proteins (VLDL) to HDL was abolished in PLTP–/– mice. On a chow diet, PLTP–/– mice showed marked decreases in HDL phospholipid (60%), cholesterol (65%), and apo AI (85%), but no significant change in non-HDL lipid or apo B levels, compared with wild-type littermates. On a high-fat diet, HDL levels were similarly decreased, but there was also an increase in VLDL and LDL phospholipids (210%), free cholesterol (60%), and cholesteryl ester (40%) without change in apo B levels, suggesting accumulation of surface components of TRL. Vesicular lipoproteins were shown by negative-stain electron microscopy of the free cholesterol– and phospholipid-enriched IDL/LDL fraction. Thus, PLTP is the major factor facilitating transfer of VLDL phospholipid into HDL. Reduced plasma PLTP activity causes markedly decreased HDL lipid and apoprotein, demonstrating the importance of transfer of surface components of TRL in the maintenance of HDL levels. Vesicular lipoproteins accumulating in PLTP–/– mice on a high-fat diet could influence the development of atherosclerosis. PMID:10079112

  1. Effect of temperature on oxidative stress, antioxidant levels and uncoupling protein expression in striped hamsters.

    PubMed

    Zhou, Si-Si; Cao, Li-Li; Xu, Wei-Dong; Cao, Jing; Zhao, Zhi-Jun

    2015-11-01

    According to the rate of living-free radical hypothesis, higher metabolic rates should increase reactive oxygen species (ROS) production. However, the "uncoupling to survive" hypothesis postulates that uncoupling proteins (UCPs) can decrease ROS production by lowering the potential of the inner mitochondrial membrane, in which case the correlation between metabolic rate and ROS levels would be a negative rather than positive. In this study, we examined energy intake, oxidative stress levels, antioxidant activity and the expression of UCPs in brown adipose tissue (BAT), and in the liver, heart, skeletal muscle and brain, of striped hamsters (Cricetulus barabensis) acclimated to either 5 °C or 32.5 °C. The energy intake of hamsters acclimated to 5 °C increased by 70.7%, whereas the energy intake of hamsters acclimated to 32.5 °C decreased by 31.3%, relative to hamsters kept at room temperature (21 °C) (P<0.05). Malonadialdehyde (MDA) levels, total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-PX) activity in BAT significantly decreased in 5 °C group, but increased in 32.5 °C group, relative to the 21 °C group. Neither ROS levels (i.e. H2O2 levels), nor antioxidants in skeletal muscle, liver, heart or brain tissue, were affected by temperature. UCP1 expression in BAT was significantly up-regulated in 5 °C group, but down-regulated in 32.5 °C group, relative to the 21 °C group. UCP3 expression of skeletal muscle was also up-regulated significantly in hamsters acclimated to 5 °C. These results suggest that the relationship between ROS levels and metabolic rate was negative, rather than positive. UCP1 expression in BAT may have played a role in lowering ROS levels.

  2. Plasma agouti-related protein levels in women with anorexia nervosa.

    PubMed

    Moriya, Junko; Takimoto, Yoshiyuki; Yoshiuchi, Kazuhiro; Shimosawa, Tatsuo; Akabayashi, Akira

    2006-10-01

    Agouti-related protein (AGRP) is the competitive antagonist of alpha-melanocyte stimulating hormone (alpha-MSH) located at melanocortin receptors 3 and 4 (MC3R and MC4R), and also acts as an MC4R inverse agonist. Hypothalamic AGRP controls food intake and body weight in rodents. It has also been found in human plasma. To study the possibility of disturbances in melanocortin receptor-related peptides in eating disorders, plasma AGRP, alpha-MSH, and leptin levels were measured in 18 female patients with anorexia nervosa (AN) (age, 23.5+/-7.1 yr; body mass index (BMI) 14.5+/-1.8 kg/m(2)) and 17 age-matched female controls (age, 25.8+/-3.9 yr; BMI 20.2+/-1.6 kg/m(2)). Blood samples were collected after overnight fasting, and plasma peptides levels were measured using ELISA. Plasma AGRP levels increased significantly in AN patients when compared with controls (P<0.01) while plasma alpha-MSH levels were not significantly different. Plasma leptin levels decreased significantly in AN patients when compared with controls (P<0.001). In addition, plasma AGRP levels were negatively correlated with leptin (r=-0.41, P<0.01) and BMI (r=-0.40, P<0.05) in all subjects. In conclusion, plasma AGRP elevation may be related to energy homeostasis disturbance in AN, and in addition to leptin, peripheral AGRP levels could be used as a nutritional marker in AN patients.

  3. The impact of dietary protein levels on nutrient digestibility and water and nitrogen balances in eventing horses.

    PubMed

    Oliveira, C A A; Azevedo, J F; Martins, J A; Barreto, M P; Silva, V P; Julliand, V; Almeida, F Q

    2015-01-01

    This study was performed to evaluate the impact of dietary protein levels on nutrient digestibility and water and nitrogen balances in conditioning eventing horses. Twenty-four Brazilian Sport Horses, male and female (8.0 to 15.0 yr; 488 ± 32 kg BW), were used in a randomized design with 4 levels of CP diets: 7.5%, 9.0%, 11.0%, and 13.0%. A digestion assay was performed with partial feces collection over 4 d, followed by 1 d of total urine collection. Data were submitted to regression analysis and adjusted to linear and quadratic models (P < 0.05). No differences were observed in the intake of DM, OM, EE, ADF, and NDF as a function of dietary protein levels. Dry matter intake average was 1.7% of BW. CP and N intake showed a linear increase as a function of increasing protein level in diets. A quadratic response (P < 0.05) was observed on the CP and NDF digestibility coefficients, with the maximum estimated level of digestibility at 11.6% and 11.4% CP in the diet, respectively. There was a linear effect on ADF digestibility coefficients, digestible DM and protein intake, and CP/DE ratio according to dietary protein levels. There was no impact of dietary protein levels on daily water intake, total water intake, or fecal water excretion. Urinary excretion values showed a linear increase in response to increased dietary protein levels, but no impact was observed on water balance, with an average of 8.4 L/d. Nitrogen intake (NI), N absorption (NA), and urinary N increased linearly as a function of increasing dietary protein levels. There was no impact of dietary protein levels on N retention (NR), with an average of 7.5 g N/d. Nitrogen retention as a percentage of NI or NA showed no significant changes in the function of dietary protein levels. There was an impact of dietary protein levels on the digestibility coefficient of CP, NDF, ADF, and digestible protein intake on conditioning eventing horses. The 11.6% CP level in the diet provided an intake of 2.25 g CP/kg BW

  4. Serum levels of protein oxidation products in patients with nickel allergy.

    PubMed

    Gangemi, Sebastiano; Ricciardi, Luisa; Minciullo, Paola Lucia; Cristani, Mariateresa; Saitta, Salvatore; Chirafisi, Joselita; Spatari, Giovanna; Santoro, Giusy; Saija, Antonella

    2009-01-01

    Nickel sensitization can not only induce allergic contact dermatitis (ACD), but also can induce an overlapping disease referred to as "systemic nickel allergy syndrome" (SNAS), characterized by urticaria/angioedema and gastrointestinal symptoms correlated to the ingestion of nickel-containing foods. This study was designed to determine if oxidative stress occurs in patients with nickel allergy. Thirty-one female patients (mean age 31.26 + 13.04 years, range 16-64 years) with confirmed nickel CD underwent oral nickel challenge because of clinically suspected SNAS; serum concentrations of protein carbonyl groups (PCGs) and nitrosylated proteins (NPs; biomarkers of oxidative stress) were measured before and after oral nickel challenge as well as in healthy female controls. Twenty-three of these 31 patients were diagnosed with SNAS because they had a positive reaction to the oral nickel challenge, and 8 patients had no reaction and therefore were classified as patients with contact nickel allergy only. Although both nickel-allergic patients and controls presented similar serum levels of PCGs, NP values in nickel-allergic patients appeared higher than in controls and tended to decrease after the challenge; furthermore, serum levels of NPs in patients affected by SNAS were higher (although not significantly) than in patients with nickel ACD only. The involvement of specific biomarkers of oxidative stress such as NPs and the lack of involvement of other biomarkers such as PCGs may help to better understand the alteration of the redox homeostasis occurring in nickel ACD and particularly in SNAS.

  5. Antibiotic free selection for the high level biosynthesis of a silk-elastin-like protein

    PubMed Central

    Barroca, Mário; Rodrigues, Paulo; Sobral, Rómulo; Costa, M. Manuela R.; Chaves, Susana R.; Machado, Raul; Casal, Margarida; Collins, Tony

    2016-01-01

    Silk-elastin-like proteins (SELPs) are a family of genetically engineered recombinant protein polymers exhibiting mechanical and biological properties suited for a wide range of applications in the biomedicine and materials fields. They are being explored as the next generation of biomaterials but low productivities and use of antibiotics during production undermine their economic viability and safety. We have developed an industrially relevant, scalable, fed-batch process for the high level production of a novel SELP in E. coli in which the commonly used antibiotic selection marker of the expression vector is exchanged for a post segregational suicide system, the separate-component-stabilisation system (SCS). SCS significantly augments SELP productivity but also enhances the product safety profile and reduces process costs by eliminating the use of antibiotics. Plasmid content increased following induction but no significant differences in plasmid levels were discerned when using SCS or the antibiotic selection markers under the controlled fed-batch conditions employed. It is suggested that the absence of competing plasmid-free cells improves host cell viability and enables increased productivity with SCS. With the process developed, 12.8 g L−1 purified SELP was obtained, this is the highest SELP productivity reported to date and clearly demonstrates the commercial viability of these promising polymers. PMID:27982135

  6. Emergence of tissue sensitivity to Hox protein levels underlies the evolution of an adaptive morphological trait

    PubMed Central

    Refki, Peter Nagui; Armisén, David; Crumière, Antonin Jean Johan; Viala, Séverine; Khila, Abderrahman

    2014-01-01

    Growth control scales morphological attributes and, therefore, provides a critical contribution to the evolution of adaptive traits. Yet, the genetic mechanisms underlying growth in the context of specific ecological adaptations are poorly understood. In water striders, adaptation to locomotion on the water surface is associated with allometric and functional changes in thoracic appendages, such that T2-legs, used as propelling oars, are longer than T3-legs, used as steering rudders. The Hox gene Ubx establishes this derived morphology by elongating T2-legs but shortening T3-legs. Using gene expression assays, RNAi knockdown, and comparative transcriptomics, we demonstrate that the evolution of water surface rowing as a novel means of locomotion is associated with the evolution of a dose-dependent promoting-repressing effect of Ubx on leg growth. In the water strider Limnoporus dissortis, T3-legs express six to seven times higher levels of Ubx compared to T2-legs. Ubx RNAi shortens T2-legs and the severity of this phenotype increases with increased depletion of Ubx protein. Conversely, Ubx RNAi lengthens T3-legs but this phenotype is partially rescued when Ubx protein is further depleted. This dose-dependent effect of Ubx on leg growth is absent in non-rowing relatives that retain the ancestral relative leg length. We also show that the spatial patterns of expression of dpp, wg, hh, egfr, dll, exd, hth, and dac are unchanged in Ubx RNAi treatments. This indicates that the dose-dependent opposite effect of Ubx on T2- and T3-legs operates without any apparent effect on the spatial expression of major leg patterning genes. Our data suggest that scaling of adaptive allometries can evolve through changes in the levels of expression of Hox proteins early during ontogeny, and in the sensitivity of the tissues that express them, without any major effects on pattern formation. PMID:24886828

  7. Heat shock proteins in relation to heat stress tolerance of creeping bentgrass at different N levels.

    PubMed

    Wang, Kehua; Zhang, Xunzhong; Goatley, Mike; Ervin, Erik

    2014-01-01

    Heat stress is a primary factor causing summer bentgrass decline. Changes in gene expression at the transcriptional and/or translational level are thought to be a fundamental mechanism in plant response to environmental stresses. Heat stress redirects protein synthesis in higher plants and results in stress protein synthesis, particularly heat shock proteins (HSPs). The goal of this work was to analyze the expression pattern of major HSPs in creeping bentgrass (Agrostis stolonifera L.) during different heat stress periods and to study the influence of nitrogen (N) on the HSP expression patterns. A growth chamber study on 'Penn-A4' creeping bentgrass subjected to 38/28°C day/night for 50 days, was conducted with four nitrate rates (no N-0, low N-2.5, medium N-7.5, and high N-12.5 kg N ha-1) applied biweekly. Visual turfgrass quality (TQ), normalized difference vegetation index (NDVI), photochemical efficiency of photosystem II (Fv/Fm), shoot electrolyte leakage (ShEL), and root viability (RV) were monitored, along with the expression pattern of HSPs. There was no difference in measured parameters between treatments until week seven, except TQ at week five. At week seven, grass at medium N had better TQ, NDVI, and Fv/Fm accompanied by lower ShEL and higher RV, suggesting a major role in improved heat tolerance. All the investigated HSPs (HSP101, HSP90, HSP70, and sHSPs) were up-regulated by heat stress. Their expression patterns indicated cooperation between different HSPs and their roles in bentgrass thermotolerance. In addition, their production seems to be resource dependent. This study could further improve our understanding about how different N levels affect bentgrass thermotolerance.

  8. Auxins differentially regulate root system architecture and cell cycle protein levels in maize seedlings.

    PubMed

    Martínez-de la Cruz, Enrique; García-Ramírez, Elpidio; Vázquez-Ramos, Jorge M; Reyes de la Cruz, Homero; López-Bucio, José

    2015-03-15

    Maize (Zea mays) root system architecture has a complex organization, with adventitious and lateral roots determining its overall absorptive capacity. To generate basic information about the earlier stages of root development, we compared the post-embryonic growth of maize seedlings germinated in water-embedded cotton beds with that of plants obtained from embryonic axes cultivated in liquid medium. In addition, the effect of four different auxins, namely indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on root architecture and levels of the heat shock protein HSP101 and the cell cycle proteins CKS1, CYCA1 and CDKA1 were analyzed. Our data show that during the first days after germination, maize seedlings develop several root types with a simultaneous and/or continuous growth. The post-embryonic root development started with the formation of the primary root (PR) and seminal scutellar roots (SSR) and then continued with the formation of adventitious crown roots (CR), brace roots (BR) and lateral roots (LR). Auxins affected root architecture in a dose-response fashion; whereas NAA and IBA mostly stimulated crown root formation, 2,4-D showed a strong repressing effect on growth. The levels of HSP101, CKS1, CYCA1 and CDKA in root and leaf tissues were differentially affected by auxins and interestingly, HSP101 registered an auxin-inducible and root specific expression pattern. Taken together, our results show the timing of early branching patterns of maize and indicate that auxins regulate root development likely through modulation of the HSP101 and cell cycle proteins.

  9. How Integrated Management Strategies Promote Protein Quality of Cotton Embryos: High Levels of Soil Available N, N Assimilation and Protein Accumulation Rate

    PubMed Central

    Yang, HongKun; Meng, YaLi; Chen, BingLin; Zhang, XingYue; Wang, YouHua; Zhao, WenQing; Zhou, ZhiGuo

    2016-01-01

    Cottonseed is widely used as a source of ruminant feed and for industrial purposes. Therefore, there is a tremendous need to improve the nutritional value of cotton embryos. In this study, a conventional management (CM) and two integrated cotton management strategies (IMS1, IMS2) were performed at two soil fertility levels to study the relationships among soil N, N assimilation, embryonic protein accumulation and protein quality. The levels of proteins, essential amino acids, and semi-essential amino acids, especially those of glutamate, lysine, and methionine, were higher in IMS1 and IMS2 embryos than in CM embryos. These changes were significantly positively correlated with the soil-available N content, glutamine synthetase activity and peak value of protein accumulation rate and were negatively correlated with the free amino acid level. These results illustrated that integrated management strategies, especially the rates and timing of N application, raise the level of soil available N, which is beneficial for N assimilation in developing cotton embryos. The protein content was limited by the rate of protein accumulation rather than by the free amino acid content. The combination of target yield fertilization, a growth-driven N application schedule, a high plant density and the seedling raising with bio-organic fertilizer can substantially improve protein quality in cotton embryos, especially at a soil with low soil organic matter and total nitrogen. PMID:27532007

  10. How Integrated Management Strategies Promote Protein Quality of Cotton Embryos: High Levels of Soil Available N, N Assimilation and Protein Accumulation Rate.

    PubMed

    Yang, HongKun; Meng, YaLi; Chen, BingLin; Zhang, XingYue; Wang, YouHua; Zhao, WenQing; Zhou, ZhiGuo

    2016-01-01

    Cottonseed is widely used as a source of ruminant feed and for industrial purposes. Therefore, there is a tremendous need to improve the nutritional value of cotton embryos. In this study, a conventional management (CM) and two integrated cotton management strategies (IMS1, IMS2) were performed at two soil fertility levels to study the relationships among soil N, N assimilation, embryonic protein accumulation and protein quality. The levels of proteins, essential amino acids, and semi-essential amino acids, especially those of glutamate, lysine, and methionine, were higher in IMS1 and IMS2 embryos than in CM embryos. These changes were significantly positively correlated with the soil-available N content, glutamine synthetase activity and peak value of protein accumulation rate and were negatively correlated with the free amino acid level. These results illustrated that integrated management strategies, especially the rates and timing of N application, raise the level of soil available N, which is beneficial for N assimilation in developing cotton embryos. The protein content was limited by the rate of protein accumulation rather than by the free amino acid content. The combination of target yield fertilization, a growth-driven N application schedule, a high plant density and the seedling raising with bio-organic fertilizer can substantially improve protein quality in cotton embryos, especially at a soil with low soil organic matter and total nitrogen.

  11. Systems level-based RNAi screening by high content analysis identifies UBR5 as a regulator of estrogen receptor-α protein levels and activity.

    PubMed

    Bolt, M J; Stossi, F; Callison, A M; Mancini, M G; Dandekar, R; Mancini, M A

    2015-01-08

    Estrogen receptor-α (ERα) is a central transcription factor that regulates mammary gland physiology and a key driver in breast cancer. In the present study, we aimed to identify novel modulators of ERα-mediated transcriptional regulation via a custom-built siRNA library screen. This screen was directed against a variety of coregulators, transcription modifiers, signaling molecules and DNA damage response proteins. By utilizing a microscopy-based, multi-end point, estrogen responsive biosensor cell line platform, the primary screen identified a wide range of factors that altered ERα protein levels, chromatin remodeling and mRNA output. We then focused on UBR5, a ubiquitin ligase and known oncogene that modulates ERα protein levels and transcriptional output. Finally, we demonstrated that UBR5 also affects endogenous ERα target genes and E2-mediated cell proliferation in breast cancer cells. In conclusion, our multi-end point RNAi screen identified novel modulators of ERα levels and activity, and provided a robust systems level view of factors involved in mechanisms of nuclear receptor action and pathophysiology. Utilizing a high throughput RNAi screening approach we identified UBR5, a protein commonly amplified in breast cancer, as a novel regulator of ERα protein levels and transcriptional activity.

  12. Different mechanical loading protocols influence serum cartilage oligomeric matrix protein levels in young healthy humans.

    PubMed

    Niehoff, A; Kersting, U G; Helling, S; Dargel, J; Maurer, J; Thevis, M; Brüggemann, G-P

    2010-10-01

    The purpose of the study was to investigate whether a relationship between the loading mode of physical activity and serum cartilage oligomeric matrix protein (COMP) concentration exists and whether the lymphatic system contributes to COMP release into the serum. Serum COMP levels were determined in healthy male subjects before, after and at 18 further time points within 7 h at four separate experimental days with four different loading interventions. The loading intervention included high impact running exercise, slow but deep knee bends, and lymphatic drainage of 30 min duration, respectively, and a resting protocol. The serum COMP levels were measured using a commercially available quantitative enzyme-linked immunosorbent assay. An increase (p < 0.001) in serum COMP concentration was detected immediately after 30 min running exercise. Slow but deep knee bends did not cause any significant changes in serum COMP levels. Lymphatic drainage also had no effect on the serum COMP concentration. After 30 min of complete rest the serum COMP level was significantly (p = 0.008) reduced. The elevation of COMP serum concentration seems to depend on the loading mode of the physical activity and to reflect the extrusion of COMP fragments from the impact loaded articular cartilage or synovial fluid.

  13. Metformin impairs systemic bile acid homeostasis through regulating SIRT1 protein levels.

    PubMed

    Chen, Qi; Yang, Xiaoying; Zhang, Huabing; Kong, Xingxing; Yao, Lu; Cui, Xiaona; Zou, Yongkang; Fang, Fude; Yang, Jichun; Chang, Yongsheng

    2017-01-01

    Metformin is widely used to treat hyperglycemia. However, metformin treatment may induce intrahepatic cholestasis and liver injury in a few patients with type II diabetes through an unknown mechanism. Here we show that metformin decreases SIRT1 protein levels in primary hepatocytes and liver. Both metformin-treated wild-type C57 mice and hepatic SIRT1-mutant mice had increased hepatic and serum bile acid levels. However, metformin failed to change systemic bile acid levels in hepatic SIRT1-mutant mice. Molecular mechanism study indicates that SIRT1 directly interacts with and deacetylates Foxa2 to inhibit its transcriptional activity on expression of genes involved in bile acids synthesis and transport. Hepatic SIRT1 mutation elevates Foxa2 acetylation levels, which promotes Foxa2 binding to and activating genes involved in bile acids metabolism, impairing hepatic and systemic bile acid homeostasis. Our data clearly suggest that hepatic SIRT1 mediates metformin effects on systemic bile acid metabolism and modulation of SIRT1 activity in liver may be an attractive approach for treatment of bile acid-related diseases such as cholestasis.

  14. C-reactive protein level and microbial aetiology in patients hospitalised with acute exacerbation of COPD.

    PubMed

    Clark, Tristan W; Medina, Marie-Jo; Batham, Sally; Curran, Martin D; Parmar, Surendra; Nicholson, Karl G

    2015-01-01

    Both viruses and bacteria are thought to cause exacerbations of chronic obstructive pulmonary disease (COPD); however, the relative importance of each remains uncertain. C-reactive protein (CRP) levels increase during exacerbations but the relationship with aetiology is not established. We aimed to explore the relationship between serum CRP and the rate of detection of viruses and bacteria. This was a prospectively recruited, observational study of patients hospitalised with exacerbations of COPD. Nasopharyngeal swabs were tested for respiratory viruses by reverse transcriptase-PCR. Sputum and blood were collected for bacterial culture and urine tested for pneumococcal antigen. CRP levels were measured on sera. CRP and other factors associated with viral, bacterial or mixed detection were assessed using multiple logistic regression analysis. 264 patients with exacerbations of COPD were studied: 26% tested positive for respiratory viruses only, 13% had bacteria only, 12% had mixed viral/bacterial detection, and 49% had no pathogens detected. CRP level and temperature were strongly associated with viral detection rate (p<0.001 and p=0.004, respectively) and mixed viral/bacterial detection rate (p=0.02 and p=0.03, respectively) on multivariate analysis. Bacterial detection rate was not associated with CRP level or body temperature. This study supports the role of viruses as important aetiological agents causing exacerbations of COPD.

  15. Increased Circulating Levels of Vitamin D Binding Protein in MS Patients

    PubMed Central

    Rinaldi, Arturo Ottavio; Sanseverino, Isabella; Purificato, Cristina; Cortese, Antonio; Mechelli, Rosella; Francisci, Silvia; Salvetti, Marco; Millefiorini, Enrico; Gessani, Sandra; Gauzzi, Maria Cristina

    2015-01-01

    Vitamin D (vitD) low status is currently considered a main environmental factor in multiple sclerosis (MS) etiology and pathogenesis. VitD and its metabolites are highly hydrophobic and circulate mostly bound to the vitamin D binding protein (DBP) and with lower affinity to albumin, while less than 1% are in a free form. The aim of this study was to investigate whether the circulating levels of either of the two vitD plasma carriers and/or their relationship are altered in MS. We measured DBP and albumin plasma levels in 28 MS patients and 24 healthy controls. MS patients were found to have higher DBP levels than healthy subjects. Concomitant interferon beta therapy did not influence DBP concentration, and the difference with the control group was significant in both females and males. No significant correlation between DBP and albumin levels was observed either in healthy controls or in patients. These observations suggest the involvement of DBP in the patho-physiology of MS. PMID:25590278

  16. Associations of High Sensitivity C-Reactive Protein Levels in Schizophrenia and Comparison Groups

    PubMed Central

    Joseph, Jamie; Depp, Colin; Martin, Averria Sirkin; Daly, Rebecca; Glorioso, Danielle K; Palmer, Barton W; Jeste, Dilip V

    2015-01-01

    Schizophrenia is characterized by physical (mainly metabolic and cardiovascular) comorbidity and shortened lifespan. High sensitivity C- reactive protein (hs-CRP), an inflammatory marker of hepatic origin linked to metabolic and cardiovascular diseases and mortality in the general population, has been reported to be elevated in people with schizophrenia. However, the relationship of hs-CRP to psychiatric and medical risk factors, after controlling for potentially confounding variables such as smoking, is not well established in schizophrenia. We assessed hs-CRP levels along with various demographic, psychiatric, and metabolic measures in 88 clinically stable outpatients with schizophrenia or schizoaffective disorder and 71 age epoch-matched comparison subjects with no history of a major psychiatric illness. hs-CRP levels were significantly higher in individuals with schizophrenia than in comparison subjects. Higher hs-CRP levels in schizophrenia group were associated with female gender, more severe negative symptoms, greater medical comorbidity, and worse metabolic risk factors including BMI, fasting glucose, and hemoglobin A1c levels. hs-CRP was not related to age, race, education, smoking status, antipsychotic dosage, or cognitive impairment. Longitudinal studies are needed to investigate the relationship between hs-CRP and long-term health outcomes including metabolic syndrome, cardiovascular disease, and mortality in schizophrenia. PMID:26341579

  17. Low-level lasers affect uncoupling protein gene expression in skin and skeletal muscle tissues

    NASA Astrophysics Data System (ADS)

    Canuto, K. S.; Sergio, L. P. S.; Paoli, F.; Mencalha, A. L.; Fonseca, A. S.

    2016-03-01

    Wavelength, frequency, power, fluence, and emission mode determine the photophysical, photochemical, and photobiological responses of biological tissues to low-level lasers. Free radicals are involved in these responses acting as second messengers in intracellular signaling processes. Irradiated cells present defenses against these chemical species to avoid unwanted effects, such as uncoupling proteins (UCPs), which are part of protective mechanisms and minimize the effects of free radical generation in mitochondria. In this work UCP2 and UCP3 mRNA gene relative expression in the skin and skeletal muscle tissues of Wistar rats exposed to low-level red and infrared lasers was evaluated. Samples of the skin and skeletal muscle tissue of Wistar rats exposed to low-level red and infrared lasers were withdrawn for total RNA extraction, cDNA synthesis, and the evaluation of gene expression by quantitative polymerase chain reaction. UCP2 and UCP3 mRNA expression was differently altered in skin and skeletal muscle tissues exposed to lasers in a wavelength-dependent effect, with the UCP3 mRNA expression dose-dependent. Alteration on UCP gene expression could be part of the biostimulation effect and is necessary to make cells exposed to red and infrared low-level lasers more resistant or capable of adapting in damaged tissues or diseases.

  18. Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level, Genotype and Developmental Stages.

    PubMed

    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 (P<0.05) gradually with increasing age. Bama mini-pigs had generally higher (P<0.05) muscle concentrations of flavor-related 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

  19. Anti-ageing properties of Khelma Longevity™: Treatment of human fibroblasts increases proteasome levels and decreases the levels of oxidized proteins.

    PubMed

    Voutetakis, Konstantinos; Delitsikou, Vasiliki; Magouritsas, Michel Georges; Gonosa, Efstathios S

    2017-03-05

    We have determined the putative anti-ageing properties of Khelma Longevity™, a formula based on various natural compounds from the Mediterranean area. Human primary fibroblast cultures were treated with a wide range of concentrations of Khelma Longevity™ for 1 day or 3 consecutive days. Following these treatments, two major and complementary biomarkers of ageing where measured, namely, the proteasome and the amount of oxidized proteins. It was observed that 24hours of treatment with Khelma Longevity™ resulted in a maximum increase of about 41% of the total protein levels of 20S proteasome. Levels of oxidized proteins were reduced by almost 6.5-fold following longer treatments. Specifically we have observed a maximum decrease of protein carbonyls to 84.7% in comparison with nontreated control cells following 3 days of continuous treatment with Khelma Longevity™. These results support the notion that formulas rich in natural compounds from the Mediterranean area possess anti-ageing properties.

  20. Carvacrol attenuates serum levels of total protein, phospholipase A2 and histamine in asthmatic guinea pig

    PubMed Central

    Boskabady, Mohammad Hossein; Jalali, Sedigheh; Yahyazadeh, Negin; Boskabady, Mostafa

    2016-01-01

    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<0.05 to p<0.001). Significant reduction in TP, PLA2 and histamine levels were observed in treated groups with the two higher concentrations of carvacrol but dexamethasone treatment only decreased serum level of PLA2 (p<0.05 to p<0.001). Although the effect of the lowest concentration of the extract was less than that of dexamethasone (p<0.05 for TP and p<0.001 for PLA2), the effects of the two higher concentrations on PLA2 were similar to dexamethasone and on TP (p<0.01) and histamine (p<0.001) were higher than those of dexamethasone. Conclusion: These results showed that carvacrol reduced serum levels of TP, PLA2 and histamine in sensitized guinea pigs which may indicate an anti-inflammatory effect of this agent in inflammatory disorders such as asthma. PMID:28078244

  1. MAP17 and SGLT1 Protein Expression Levels as Prognostic Markers for Cervical Tumor Patient Survival

    PubMed Central

    Perez, Marco; Praena-Fernandez, Juan M.; Felipe-Abrio, Blanca; Lopez-Garcia, Maria A.; Lucena-Cacace, Antonio; Garcia, Angel; Lleonart, Matilde; Roncador, Guiovanna; Marin, Juan J.; Carnero, Amancio

    2013-01-01

    MAP17 is a membrane-associated protein that is overexpressed in human tumors. Because the expression of MAP17 increases reactive oxygen species (ROS) generation through SGLT1 in cancer cells, in the present work, we investigated whether MAP17 and/or SGLT1 might be markers for the activity of treatments involving oxidative stress, such as cisplatin or radiotherapy. First, we confirmed transcriptional alterations in genes involved in the oxidative stress induced by MAP17 expression in HeLa cervical tumor cells and found that Hela cells expressing MAP17 were more sensitive to therapies that induce ROS than were parental cells. Furthermore, MAP17 increased glucose uptake through SGLT receptors. We then analyzed MAP17 and SGLT1 expression levels in cervical tumors treated with cisplatin plus radiotherapy and correlated the expression levels with patient survival. MAP17 and SGLT1 were expressed in approximately 70% and 50% of cervical tumors of different types, respectively, but they were not expressed in adenoma tumors. Furthermore, there was a significant correlation between MAP17 and SGLT1 expression levels. High levels of either MAP17 or SGLT1 correlated with improved patient survival after treatment. However, the patients with high levels of both MAP17 and SGLT1 survived through the end of this study. Therefore, the combination of high MAP17 and SGLT1 levels is a marker for good prognosis in patients with cervical tumors after cisplatin plus radiotherapy treatment. These results also suggest that the use of MAP17 and SGLT1 markers may identify patients who are likely to exhibit a better response to treatments that boost oxidative stress in other cancer types. PMID:23418532

  2. Are serum eosinophilic cationic protein levels of toll collectors affected by diesel exhaust exposure?

    PubMed Central

    Bilgin, Cahit; Arbak, Peri; Yavuz, Ozlem; Balbay, Ege Gulec; Balbay, Oner; Annakkaya, Ali Nihat

    2016-01-01

    Objective: There are few studies on the diesel exhaust particulates (DEP) / eosinophilic cationic protein (ECP) level relationship. This study aimed to detect ECP levels in a highly DE exposed group, named as toll collectors. Methods: In a cross-sectional study, levels of serum ECP, rates of respiratory symptoms, mean levels of respiratory functions, smoking status, and variations in peak expiratory flow (PEF) during weekends and working days were compared for 68 toll collectors (TC) (range of age, 24-48 years) and 28 controls (range of age, 25-61 years). All subjects in the study group were men. Results: No significant difference was observed in terms of symptoms and smoking rates between the toll collectors and control group. The number of toll collectors [12/68 (17.7%) vs 1/28 (3.5%)] with diurnal PEF variability in the working period was higher than that of controls (p=0.058). Mean ECP level of toll collectors was higher than that of controls (32.8 vs 21.4 ng/L), but the difference was not significant. Mean ECP levels were higher in subjects experiencing diurnal PEF variability during work and off-work periods (34.9 vs 28.3 ng/L, p=0.410). Conclusions: Serial PEF measurements combined with serum ECP measurements did not add a new tool to detect the sensitivity of workers dealing with DE. Much more diesel exhaust exposed workers should be included to search for cheap and available methods when evaluating airway. PMID:27882015

  3. Effects of Different Exercise Intensities with Isoenergetic Expenditures on C-Reactive Protein and Blood Lipid Levels

    ERIC Educational Resources Information Center

    Tsao, Te Hung; Yang, Chang Bin; Hsu, Chin Hsing

    2012-01-01

    We investigated the effects of different exercise intensities on C-reactive protein (CRP), and whether changes in CRP levels correlated with blood lipid levels. Ten men exercised at 25%, 65%, and 85% of their maximum oxygen consumption rates. Participants' blood was analyzed for CRP and blood lipid levels before and after the exercise sessions.…

  4. CSF levels of heart fatty acid binding protein are altered during early phases of Alzheimer's disease.

    PubMed

    Chiasserini, Davide; Parnetti, Lucilla; Andreasson, Ulf; Zetterberg, Henrik; Giannandrea, David; Calabresi, Paolo; Blennow, Kaj

    2010-01-01

    Heart fatty acid binding protein (HFABP) has been proposed as a putative marker for dementia disorders. To evaluate the value of this protein as an early marker of Alzheimer's disease (AD), we analyzed HFABP level and the classical biomarkers amyloid-β (Aβ)1-42, total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF) of patients with mild cognitive impairment (MCI) followed up for four years (n=41), AD (n=32), and subjects with other neurological diseases without dementia (OND, n=25). HFABP levels were higher in AD patients and in MCI converting to AD (MCI-AD) with respect to OND and to cognitively stable MCI patients (MCI-MCI). The receiver operator characteristics analysis for HFABP alone showed a sensitivity of 87% and a specificity of 81% for AD versus OND (area under the curve, AUC=0.83); sensitivity and specificity were 46% and 94%, respectively, when comparing MCI-MCI versus MCI-AD. CSF HFABP levels showed a strong positive correlation with both t-tau and p-tau. Interestingly, the ratio between HFABP and Aβ1-42 improved the performance in distinguishing AD from OND (sensitivity: 90%; specificity 82%, AUC=0.89), and gave the best accuracy in discriminating MCI-AD from MCI-MCI (sensitivity: 80%; specificity 100%, AUC=0.90). Survival analysis by means of Kaplan-Meier curve showed a significantly higher proportion of MCI patients converting to AD in the group with higher values of HFABP/Aβ1-42 ratio (cut-off=0.7). A significant correlation between HFABP/Aβ1-42 ratio and MMSE annual decrease rate was also documented (p<0.0001). HFABP /Aβ1-42 ratio might be a useful predictor of conversion in MCI patients.

  5. Matrix Gla protein (MGP) promoter polymorphic variants and its serum level in stenosis of coronary artery.

    PubMed

    Najafi, Mohammad; Roustazadeh, Abazar; Amirfarhangi, Abdollah; Kazemi, Bahram

    2014-03-01

    Although the role of matrix Gla protein (MGP) is not completely known but, its expression within subendothelial macrophages and vascular smooth muscle cells is suggested to be involved in vascular calcification. In this study, we investigated the associations between the serum MGP levels and the MGP promoter high minor allele frequency (MAF) variants with the development of stenosis in coronary arteries. Moreover, we evaluated the allele changes within predicted transcription factor elements with bioinformatics tools. 182 subjects were recruited from who underwent coronary angiography. The MGP promoter rs1800801, rs1800802 and rs1800799 genotypes and haplotypes were detected by ARMS-RFLP PCR techniques. The serum MGP concentration was measured using ELISA method. Jaspar profiles were used for scoring the polymorphic variations within the transcription factor elements. The genotype and two-allelic haplotype distributions were not significant between the patient and control groups (P > 0.05). The serum MGP levels had not significant differences between the genotypes (P > 0.1) and haplotypes (P > 0.4). Based on the prediction studies, we did not observe significant differences between the polymorphic scores in the predicted elements (P > 0.05). We concluded that the genotype and haplotype distributions of the MGP promoter high-MAF polymorphisms, as confirmed in the prediction studies and the serum MGP level are not significantly associated with the coronary artery disease. Based on the study results, the MGP protein did not play an important role in the development of stenosis of coronary arteries.

  6. Fish protein intake induces fast-muscle hypertrophy and reduces liver lipids and serum glucose levels in rats.

    PubMed

    Kawabata, Fuminori; Mizushige, Takafumi; Uozumi, Keisuke; Hayamizu, Kohsuke; Han, Li; Tsuji, Tomoko; Kishida, Taro

    2015-01-01

    In our previous study, fish protein was proven to reduce serum lipids and body fat accumulation by skeletal muscle hypertrophy and enhancing basal energy expenditure in rats. In the present study, we examined the precise effects of fish protein intake on different skeletal muscle fiber types and metabolic gene expression of the muscle. Fish protein increased fast-twitch muscle weight, reduced liver triglycerides and serum glucose levels, compared with the casein diet after 6 or 8 weeks of feeding. Furthermore, fish protein upregulated the gene expressions of a fast-twitch muscle-type marker and a glucose transporter in the muscle. These results suggest that fish protein induces fast-muscle hypertrophy, and the enhancement of basal energy expenditure by muscle hypertrophy and the increase in muscle glucose uptake reduced liver lipids and serum glucose levels. The present results also imply that fish protein intake causes a slow-to-fast shift in muscle fiber type.

  7. Effect of dietary protein quality and feeding level on milk secretion and mammary protein synthesis in the rat

    SciTech Connect

    Sampson, D.A.; Jansen, G.R.

    1985-04-01

    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-/sup 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.

  8. The effect of dietary energy and protein levels on production in breeding female ostriches.

    PubMed

    Brand, Z; Brand, T S; Brown, C R

    2003-09-01

    1. In a study spanning two breeding seasons, we assessed the effect of different dietary energy and protein levels on body mass, body condition, and egg production of female ostriches. 2. During the first breeding season, groups were given diets with energy concentrations of 8.5, 9.5 and 10.5 MJ/kg dry mass (DM) metabolisable energy (ME) and protein concentrations of 135, 150 and 165 g/kg. In the second breeding season, groups were given diets with ME of 7.5, 8.5 and 9.5 MJ/kg and protein contents of 105, 120 and 135 g/kg. 3. Body mass of birds on diets of 7.5 and 8.5 MJ/kg ME decreased significantly in the course of the breeding season compared with birds fed on diets with higher energy contents and body measurements decreased, suggesting a loss of body condition. 4. Females fed on diets containing only 7.5 MJ/kg ME produced significantly fewer eggs at significantly longer intervals, resulting in fewer chicks hatched. 5. There was no significant difference in egg mass, initial chick mass, chick survival to one month of age and body mass of chicks at one month. 6. Dietary protein concentrations had no effect on egg production, egg mass, hatchability, initial chick mass, chick survival or chick mass at one month old. 7. The female ostriches regained their original body mass during the 4-month rest period between breeding seasons, but significant differences in some parameters during the second breeding season suggest that they may not have fully recovered their body condition. 8. A dietary energy content of 7.5 MJ/kg proved to have an adverse effect on egg production by breeding female ostriches, and it may be concluded from this study that a diet containing 8.5 MJ ME/kg DM and 105 g/kg protein should be regarded as the minimum that can be used for breeding female ostriches without compromising egg production.

  9. Genetic Markers Associated with Plasma Protein C Level in African Americans: the Atherosclerosis Risk in Communities (ARIC) Study

    PubMed Central

    Munir, M. Shahzeb; Weng, Lu-Chen; Tang, Weihong; Basu, Saonli; Pankow, James S.; Matijevic, Nena; Cushman, Mary; Boerwinkle, Eric; Folsom, Aaron R.

    2015-01-01

    Protein C is an endogenous anticoagulant protein with anti-inflammatory properties. Single-nucleotide polymorphisms (SNPs) affect the levels of circulating protein C in European Americans. We performed a genome-wide association (GWA) scan of plasma protein C concentration with approximately 2.5 million SNPs in 2,701 African Americans in the Atherosclerosis Risk in Communities Study. Seventy-nine SNPs from the 20q11 and 2q14 regions reached the genome-wide significance threshold of 5 × 10−8. A missense variant rs867186 in the PROCR gene at 20q11 is known to affect protein C levels in individuals of European descent and showed the strongest signal (P = 9.84 × 10−65) in African Americans. The minor allele of this SNP was associated with higher protein C levels (β = 0.49 μg/ml; 10% variance explained). In the 2q14 region, the top SNPs were near or within the PROC gene: rs7580658 (β = 0.15 μg/ml; 2% variance explained, P = 1.7 × 10−12) and rs1799808 (β = 0.15 μg/ml; 2% variance explained, P = 2.03 × 10−12). These two SNPs were in strong linkage disequilibrium (LD) with another SNP rs1158867 that resides in a biochemically functional site and in weak to strong LD with the top PROC variants previously reported in individuals of European descent. In addition, two variants outside the PROC region were significantly and independently associated with protein C levels: rs4321325 in CYP27C1 and rs13419716 in MYO7B. In summary, this first GWA study for plasma protein C levels in African Americans confirms the associations of SNPs in the PROC and PROCR regions with circulating levels of protein C across ethnic populations and identifies new candidates for protein C regulation. PMID:25376901

  10. Effects of Dietary Crude Protein Levels and Cysteamine Supplementation on Protein Synthetic and Degradative Signaling in Skeletal Muscle of Finishing Pigs

    PubMed Central

    Zhou, Ping; Zhang, Lin; Li, Jiaolong; Luo, Yiqiu; Zhang, Bolin; Xing, Shen; Zhu, Yuping; Sun, Hui; Gao, Feng; Zhou, Guanghong

    2015-01-01

    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

  11. Effects of Dietary Crude Protein Levels and Cysteamine Supplementation on Protein Synthetic and Degradative Signaling in Skeletal Muscle of Finishing Pigs.

    PubMed

    Zhou, Ping; Zhang, Lin; Li, Jiaolong; Luo, Yiqiu; Zhang, Bolin; Xing, Shen; Zhu, Yuping; Sun, Hui; Gao, Feng; Zhou, Guanghong

    2015-01-01

    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

  12. Eosinophil cationic protein serum levels and allergy in chronic fatigue syndrome.

    PubMed

    Conti, F; Magrini, L; Priori, R; Valesini, G; Bonini, S

    1996-02-01

    Chronic fatigue syndrome (CFS) is a syndrome of uncertain etiopathogenesis characterized by disabling fatigue associated with a variable number of somatic and/or neuropsychologic symptoms. In patients with CFS, several immunologic abnormalities can be detected, including a higher prevalance of allergy. The aim of this study was to determine whether CFS patients, well studied for their allergy profile, show signs of eosinophil activation, as detectable by the measurement in serum of eosinophil cationic protein (ECP) levels. In 35 consecutive CFS outpatients (diagnosis based on the Centers for Disease Control case definition), ECP was measured in serum by a competitive enzyme immunoassay (ECP-FEIA kit, Kabi Pharmacia Diagnostics, Uppsala, Sweden). Fourteen disease-free subjects with no history of CFS or allergy were selected as controls. ECP serum levels were significantly higher in CFS patients than in controls (18.0 +/- 11.3 micrograms/l vs 7.3 +/- 2.1 micrograms/l; P < 0.01). In the CFS population, the prevalence of RAST positivity to one or more allergens was 77%, while no control showed positive RAST. Twelve of the 14 CFS patients with increased ECP serum levels were RAST-positive. However, CFS RAST-positive patients had no significantly higher ECP serum levels than CFS RAST-negative patients (19.3 +/- 12.4 micrograms/l vs 13.6 +/- 3.7 micrograms/l; P = 0.4). This is the first report of increased serum levels of ECP in CFS. On the basis of the available data, it is discussed whether eosinophil activation has a pathogenetic role in CFS or is linked to the frequently associated allergic condition, or, finally, whether a common immunologic background may exist for both atopy and CFS.

  13. Hypoxia and DNA-damaging agent bleomycin both increase the cellular level of the protein 4E-BP.

    PubMed

    Le Bouffant, Ronan; Cormier, Patrick; Mulner-Lorillon, Odile; Bellé, Robert

    2006-09-01

    The 4E-binding proteins (4E-BPs) regulate the cap-dependent eukaryotic initiation factor 4E (eIF4E). The level of 4E-BP protein is regulated during early development of sea urchin embryos. Fertilization leads to the rapid disappearance of the protein that reappears later in development. We show that two important cellular stresses, hypoxia and bleomycin prolonged checkpoint mobilization provoked the overexpression of the protein 4E-BP in developing sea urchin embryos. Hypoxia resulted after 1 h in a reversible gradual increase in the protein 4E-BP level. At 20 h, the protein 4E-BP had reached the level existing in the unfertilized eggs. Bleomycin used as a DNA-damaging agent for checkpoint activation, provoked cell cycle inhibition and after prolonged exposure (20 h), induced the expression of the protein 4E-BP. The effect of bleomycin on 4E-BP protein overexpression was dose-dependent between 0.4 and 1.2 mM. The role of the overexpression of the protein 4E-BP is discussed in relation with cellular stress responses.

  14. SET overexpression in HEK293 cells regulates mitochondrial uncoupling proteins levels within a mitochondrial fission/reduced autophagic flux scenario.

    PubMed

    Almeida, Luciana O; Goto, Renata N; Neto, Marinaldo P C; Sousa, Lucas O; Curti, Carlos; Leopoldino, Andréia M

    2015-03-06

    We hypothesized that SET, a protein accumulated in some cancer types and Alzheimer disease, is involved in cell death through mitochondrial mechanisms. We addressed the mRNA and protein levels of the mitochondrial uncoupling proteins UCP1, UCP2 and UCP3 (S and L isoforms) by quantitative real-time PCR and immunofluorescence as well as other mitochondrial involvements, in HEK293 cells overexpressing the SET protein (HEK293/SET), either in the presence or absence of oxidative stress induced by the pro-oxidant t-butyl hydroperoxide (t-BHP). SET overexpression in HEK293 cells decreased UCP1 and increased UCP2 and UCP3 (S/L) mRNA and protein levels, whilst also preventing lipid peroxidation and decreasing the content of cellular ATP. SET overexpression also (i) decreased the area of mitochondria and increased the number of organelles and lysosomes, (ii) increased mitochondrial fission, as demonstrated by increased FIS1 mRNA and FIS-1 protein levels, an apparent accumulation of DRP-1 protein, and an increase in the VDAC protein level, and (iii) reduced autophagic flux, as demonstrated by a decrease in LC3B lipidation (LC3B-II) in the presence of chloroquine. Therefore, SET overexpression in HEK293 cells promotes mitochondrial fission and reduces autophagic flux in apparent association with up-regulation of UCP2 and UCP3; this implies a potential involvement in cellular processes that are deregulated such as in Alzheimer's disease and cancer.

  15. Levels of synthesis of primate-specific nuclear proteins differ between growth-arrested and proliferating cells

    SciTech Connect

    Celis, J.E.; Madsen, P.; Nielsen, S.; Ratz, G.P.; Lauridsen, J.B.; Celis, A.

    1987-02-01

    A monoclonal antibody that reacts specifically with the proliferation-sensitive nuclear proteins, isoelectric focusing (IEF) 8Z31 (molecular weight (MW), 76,000 charge variants, HeLa protein catalogue number) has been characterized. As determined by indirect immunofluorescence, the antibody stains the nucleolus and nucleoplasm of interphase-cultured cells of primate origin, but does not react with cells of other species. Proteins having similar MWs and isoelectric points as the human or monkey (primates) proteins were not observed in cultured cells of the following species: aves, bat, dog, dolphin, goat, hamster, mink, mouse, pisces, potoroo, rabbit and rat. Quantitative two-dimensional (2D) gel electrophoretic analysis of (/sup 35/S)methionine-labelled proteins synthesized by normal (quiescent, proliferating) and SV40-transformed human MRC-5 fibroblasts revealed significant differences in the levels of synthesis of both IEF 8Z30 and 8Z31. In quiescent cells the main labelled product corresponded to IEF 8Z31 (ratio IEF 8Z31/8Z30, 2.3), while in the transformed cells the major product was IEF 8Z30 (ratio, 0.62). Normal proliferating fibroblasts exhibited similar levels of both proteins (ratio, 1.21). Combined levels of synthesis of both proteins were 1.50 and 1.20 times as high in the transformed cells as in the quiescent and proliferating cells, respectively. Modulation of the levels of synthesis of these proteins may play a role in cell proliferation.

  16. A comparison of ghrelin, glucose, alpha-amylase and protein levels in saliva from diabetics.

    PubMed

    Aydin, Suleyman

    2007-01-31

    During the past decade, many salivary parameters have been used to characterize disease states. Ghrelin (GAH) is recently-discovered peptide hormone secreted mainly from the stomach but also produced in a number of other tissues including salivary glands. The aim of this work was to examine the relationship between active (aGAH) and inactive (dGAH) ghrelin in the saliva and other salivary parameters in type II diabetic patients and healthy controls. Salivary parameters were assessed in a single measurement of unstimulated whole saliva from 20 obese and 20 non-obese type II diabetes patients, and in 22 healthy controls. Total protein and alpha-amylase were determined by colorimetric methods, and glucose by the glucose-oxidase method. Saliva aGAH and dGAH levels were measured using a commercial radioimmunoassay (RIA) kit. Salivary concentrations of aGAH and dGAH ghrelin were more markedly decreased in obese diabetic subjects than in the two other groups. Glucose and alpha-amylase levels were higher in diabetic subjects than in controls. Furthermore, there were correlations between GAH levels and BMI, and between GAH and blood pressure. However, there was no marked variability in saliva flow rates among the groups. These results indicate that measurement of salivary GAH and its relationship to other salivary parameters might help to provide insight into the role of ghrelin in diabetes.

  17. Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization.

    PubMed

    Miele, Claudia; Paturzo, Flora; Teperino, Raffaele; Sakane, Fumio; Fiory, Francesca; Oriente, Francesco; Ungaro, Paola; Valentino, Rossella; Beguinot, Francesco; Formisano, Pietro

    2007-11-02

    Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-alpha (PKCalpha) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms alpha and delta, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKdelta, but not of DGKalpha expression, was sufficient to prevent the effect of high glucose on PKCalpha activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCalpha activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism.

  18. The human BDNF gene: peripheral gene expression and protein levels as biomarkers for psychiatric disorders

    PubMed Central

    Cattaneo, A; Cattane, N; Begni, V; Pariante, C M; Riva, M A

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) regulates the survival and growth of neurons, and influences synaptic efficiency and plasticity. The human BDNF gene consists of 11 exons, and distinct BDNF transcripts are produced through the use of alternative promoters and splicing events. The majority of the BDNF transcripts can be detected not only in the brain but also in the blood cells, although no study has yet investigated the differential expression of BDNF transcripts at the peripheral level. This review provides a description of the human BDNF gene structure as well as a summary of clinical and preclinical evidence supporting the role of BDNF in the pathogenesis of psychiatric disorders. We will discuss several mechanisms as possibly underlying BDNF modulation, including epigenetic mechanisms. We will also discuss the potential use of peripheral BDNF as a biomarker for psychiatric disorders, focusing on the factors that can influence BDNF gene expression and protein levels. Within this context, we have also characterized, for we believe the first time, the expression of BDNF transcripts in the blood, with the aim to provide novel insights into the molecular mechanisms and signaling that may regulate peripheral BDNF gene expression levels. PMID:27874848

  19. Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication

    PubMed Central

    Naik, Anar; Decker, Markus; O’Connell, Kevin F.

    2017-01-01

    In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1, is required for centriole duplication, but our understanding of how ZYG-1 levels are regulated remains incomplete. We have identified the two PP1 orthologs, GSP-1 and GSP-2, and their regulators I-2SZY-2 and SDS-22 as key regulators of ZYG-1 protein levels. We find that down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and our data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. Our results thus define a new pathway that limits the number of daughter centrioles produced each cycle. PMID:28103229

  20. RNA-Binding Protein AUF1 Promotes Myogenesis by Regulating MEF2C Expression Levels

    PubMed Central

    Panda, Amaresh C.; Abdelmohsen, Kotb; Yoon, Je-Hyun; Martindale, Jennifer L.; Yang, Xiaoling; Curtis, Jessica; Mercken, Evi M.; Chenette, Devon M.; Zhang, Yongqing; Schneider, Robert J.; Becker, Kevin G.; de Cabo, Rafael

    2014-01-01

    The mammalian RNA-binding protein AUF1 (AU-binding factor 1, also known as heterogeneous nuclear ribonucleoprotein D [hnRNP D]) binds to numerous mRNAs and influences their posttranscriptional fate. Given that many AUF1 target mRNAs encode muscle-specific factors, we investigated the function of AUF1 in skeletal muscle differentiation. In mouse C2C12 myocytes, where AUF1 levels rise at the onset of myogenesis and remain elevated throughout myocyte differentiation into myotubes, RNP immunoprecipitation (RIP) analysis indicated that AUF1 binds prominently to Mef2c (myocyte enhancer factor 2c) mRNA, which encodes the key myogenic transcription factor MEF2C. By performing mRNA half-life measurements and polysome distribution analysis, we found that AUF1 associated with the 3′ untranslated region (UTR) of Mef2c mRNA and promoted MEF2C translation without affecting Mef2c mRNA stability. In addition, AUF1 promoted Mef2c gene transcription via a lesser-known role of AUF1 in transcriptional regulation. Importantly, lowering AUF1 delayed myogenesis, while ectopically restoring MEF2C expression levels partially rescued the impairment of myogenesis seen after reducing AUF1 levels. We propose that MEF2C is a key effector of the myogenesis program promoted by AUF1. PMID:24891619

  1. Restriction of immunoglobulin heterogeneity, autoimmunity and serum protein levels in aged people.

    PubMed Central

    Riesen, W; Keller, H; Skvaril, F; Morell, A; Barandun, S

    1976-01-01

    Ninety-one sera of persons above 80 years of age were screened for autoantibody activity against lipoproteins (anti-LDL 7, anti-HDL 6 positive), for rheumatoid factor activity (Latex 14, Waaler-Rose 7 positive) and for antinuclear factors (11 positive). Among the sera with autoantibody activity 29 percent showed deviations of the normal kappa/lambda ratio of immunoglobulins, as opposed to 22 percent of the sera without detected autoantibody activity. In 3 percent of the sera an M component was detected. Determination of the alpha1-acid glycoprotein, alpha1-antitrypsin, haptoglobin, haemopexin, complement component C3c and C4, IgG, IgA and IgM levels showed significant increases in alpha-, and beta-globulins as well as in IgG and IgA in sera of the aged persons as compared to a normal population between 20 and 60 years old. No significant difference was noted between the gamma-globulin concentration in sera of aged persons with or without autoantibody activity. The evaluation of the relationship between serum protein levels and alterations of the kappa/lambda ratio indicated that the alpha- and the beta-globulins were significantly raised in sera with altered kappa/lambda ratios, whereas, with the exception of M component containing sera the gamma-globulin levels seemed not significantly affected by changes in this ratio. PMID:62632

  2. Interpersonal Violence, PTSD, and Inflammation: Potential Psychogenic Pathways to Higher C-reactive Protein Levels

    PubMed Central

    Heath, Nicole M.; Chesney, Samantha A.; Gerhart, James I.; Goldsmith, Rachel E.; Luborsky, Judith L.; Stevens, Natalie R.; Hobfoll, Steven E.

    2013-01-01

    Interpersonal violence (IPV) is major public health concern with wide-ranging sequelae including depression, posttraumatic stress disorder (PTSD), and possible alterations of immune and inflammation processes. There is a need to identify the psycho-biological pathways through which IPV may translate to altered inflammatory processes since both PTSD and inflammation are associated with serious physical health conditions such as obesity, diabetes, and cardiovascular disease. This study investigated the relationships between IPV, psychological distress, and the inflammatory marker C-reactive protein (CRP), in a sample of 139 urban women who have a high likelihood for having experienced IPV. Participants were recruited from an outpatient gynecology clinic to complete self-report measures about their IPV histories and psychological symptoms, as well as to have their blood sampled using a finger stick. Results indicated that exposure to IPV predicted the presence of probable depression and PTSD diagnoses. Individuals who experience clinical levels of PTSD exhibited higher CRP levels, and this relationship held after adjusting for comorbid depression. Correlational analyses suggested that reexperiencing symptoms may explain the link between PTSD diagnosis and higher levels of CRP. Follow-up path analytic models provided good fit to the overall data, and indicated that the relationship between probable PTSD status and CRP is not explained by higher BMI. Overall, these findings call for increased attention to the role of PTSD in explaining links between trauma and diminished health. PMID:23701836

  3. Dynamic regulation of basement membrane protein levels promotes egg chamber elongation in Drosophila

    PubMed Central

    Isabella, Adam J.; Horne-Badovinac, Sally

    2015-01-01

    Basement membranes (BMs) are sheet-like extracellular matrices that provide essential support to epithelial tissues. Recent evidence suggests that regulated changes in BM architecture can direct tissue morphogenesis, but the mechanisms by which cells remodel BMs are largely unknown. The Drosophila egg chamber is an organ-like structure that transforms from a spherical to an ellipsoidal shape as it matures. This elongation coincides with a stage-specific increase in Type IV Collagen (Col IV) levels in the BM surrounding the egg chamber; however, the mechanisms and morphogenetic relevance of this remodeling event have not been established. Here, we identify the Collagen-binding protein SPARC as a negative regulator of egg chamber elongation, and show that SPARC down-regulation is necessary for the increase in Col IV levels to occur. We find that SPARC interacts with Col IV prior to secretion and propose that, through this interaction, SPARC blocks the incorporation of newly synthesized Col IV into the BM. We additionally observe a decrease in Perlecan levels during elongation, and show that Perlecan is a negative regulator of this process. These data provide mechanistic insight into SPARC’s conserved role in matrix dynamics and demonstrate that regulated changes in BM composition influence organ morphogenesis. PMID:26348027

  4. Reduced levels of protein recoding by A-to-I RNA editing in Alzheimer's disease

    PubMed Central

    Khermesh, Khen; D'Erchia, Anna Maria; Barak, Michal; Annese, Anita; Wachtel, Chaim; Levanon, Erez Y.; Picardi, Ernesto; Eisenberg, Eli

    2016-01-01

    Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer's disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients’ brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing. PMID:26655226

  5. Cardiorespiratory fitness, pulmonary function and C-reactive protein levels in nonsmoking individuals with diabetes

    PubMed Central

    Francisco, C.O.; Catai, A.M.; Moura-Tonello, S.C.G.; Lopes, S.L.B.; Benze, B.G.; Del Vale, A.M.; Leal, A.M.O.

    2014-01-01

    The objective of this study was to evaluate cardiorespiratory fitness and pulmonary function and the relationship with metabolic variables and C-reactive protein (CRP) plasma levels in individuals with diabetes mellitus (DM). Nineteen men with diabetes and 19 age- and gender-matched control subjects were studied. All individuals were given incremental cardiopulmonary exercise and pulmonary function tests. In the exercise test, maximal workload (158.3±22.3 vs 135.1±25.2, P=0.005), peak heart rate (HRpeak: 149±12 vs 139±10, P=0.009), peak oxygen uptake (VO2peak: 24.2±3.2 vs 18.9±2.8, P<0.001), and anaerobic threshold (VO2VT: 14.1±3.4 vs 12.2±2.2, P=0.04) were significantly lower in individuals with diabetes than in control subjects. Pulmonary function test parameters, blood pressure, lipid profile (triglycerides, HDL, LDL, and total cholesterol), and CRP plasma levels were not different in control subjects and individuals with DM. No correlations were observed between hemoglobin A1C (HbA1c), CRP and pulmonary function test and cardiopulmonary exercise test performance. In conclusion, the results demonstrate that nonsmoking individuals with DM have decreased cardiorespiratory fitness that is not correlated with resting pulmonary function parameters, HbA1c, and CRP plasma levels. PMID:24760118

  6. Cardiorespiratory fitness, pulmonary function and C-reactive protein levels in nonsmoking individuals with diabetes.

    PubMed

    Francisco, C O; Catai, A M; Moura-Tonello, S C G; Lopes, S L B; Benze, B G; Del Vale, A M; Leal, A M O

    2014-05-01

    The objective of this study was to evaluate cardiorespiratory fitness and pulmonary function and the relationship with metabolic variables and C-reactive protein (CRP) plasma levels in individuals with diabetes mellitus (DM). Nineteen men with diabetes and 19 age- and gender-matched control subjects were studied. All individuals were given incremental cardiopulmonary exercise and pulmonary function tests. In the exercise test, maximal workload (158.3 ± 22.3 vs 135.1 ± 25.2, P=0.005), peak heart rate (HRpeak: 149 ± 12 vs 139 ± 10, P=0.009), peak oxygen uptake (VO2peak: 24.2 ± 3.2 vs 18.9 ± 2.8, P<0.001), and anaerobic threshold (VO2VT: 14.1 ± 3.4 vs 12.2 ± 2.2, P=0.04) were significantly lower in individuals with diabetes than in control subjects. Pulmonary function test parameters, blood pressure, lipid profile (triglycerides, HDL, LDL, and total cholesterol), and CRP plasma levels were not different in control subjects and individuals with DM. No correlations were observed between hemoglobin A1C (HbA1c), CRP and pulmonary function test and cardiopulmonary exercise test performance. In conclusion, the results demonstrate that nonsmoking individuals with DM have decreased cardiorespiratory fitness that is not correlated with resting pulmonary function parameters, HbA1c, and CRP plasma levels.

  7. Levels of heavy metals and their binding protein metallothionein in type 2 diabetics with kidney disease.

    PubMed

    Raudenska, Martina; Dvorakova, Veronika; Pacal, Lukas; Chalasova, Katarina; Kratochvilova, Monika; Gumulec, Jaromir; Ruttkay-Nedecky, Branislav; Zitka, Ondrej; Kankova, Katerina; Adam, Vojtech; Masarik, Michal

    2017-01-06

    Hyperglycemia, a major metabolic disturbance present in diabetes, promotes oxidative stress. Activation of antioxidant defense is an important mechanism to prevent cell damage. Levels of heavy metals and their binding proteins can contribute to oxidative stress. Antiradical capacity and levels of metallothionein (MT), metals (zinc and copper), and selected antioxidants (bilirubin, cysteine, and glutathione) were determined in 70 type 2 diabetes mellitus (T2DM) subjects and 80 healthy subjects of Caucasian origin. Single nucleotide polymorphism (rs28366003) in MT gene was detected. Antiradical capacity, conjugated bilirubin, and copper were significantly increased in diabetics, whereas MT and glutathione were decreased. Genotype AA of rs28366003 was associated with higher zinc levels in the diabetic group. The studied parameters were not influenced by renal function. This is the first study comprehensively investigating differences in MT and metals relevant to oxidative stress in T2DM. Ascertained differences indicate increased oxidative stress in T2DM accompanied by abnormalities in non-enzymatic antioxidant defense systems.

  8. Ubiquitin-specific peptidase 48 regulates Mdm2 protein levels independent of its deubiquitinase activity

    PubMed Central

    Cetkovská, Kateřina; Šustová, Hana; Uldrijan, Stjepan

    2017-01-01

    The overexpression of Mdm2 has been linked to the loss of p53 tumour suppressor activity in several human cancers. Here, we present results suggesting that ubiquitin-specific peptidase 48 (USP48), a deubiquitinase that has been linked in previous reports to the NF-κB signaling pathway, is a novel Mdm2 binding partner that promotes Mdm2 stability and enhances Mdm2-mediated p53 ubiquitination and degradation. In contrast to other deubiquitinating enzymes (DUBs) that have been previously implicated in the regulation of Mdm2 protein stability, USP48 did not induce Mdm2 stabilization by significantly reducing Mdm2 ubiquitination levels. Moreover, two previously characterized USP48 mutants lacking deubiquitinase activity were also capable of efficiently stabilizing Mdm2, indicating that USP48 utilizes a non-canonical, deubiquitination-independent mechanism to promote Mdm2 oncoprotein stability. This study represents, to the best of our knowledge, the first report suggesting DUB-mediated target protein stabilization that is independent of its deubiquitinase activity. In addition, our results suggest that USP48 might represent a new mechanism of crosstalk between the NF-κB and p53 stress response pathways. PMID:28233861

  9. Protein carbonyl levels correlate with performance in elite field hockey players.

    PubMed

    Rosa-Lima, Frederico Luis; Lannes, Luiz; Viana-Gomes, Diego; Pierucci, Anna Paola T R; Salerno, Verônica P

    2015-07-01

    Excess and incorrectly selected exercise can degrade athletic performance from an imbalance in redox homeostasis and oxidative stress, but well-planned training and nutrition can improve antioxidant capacity. The aim of the study was to investigate how nutrient intake could influence oxidative stress and cell lesion biomarkers after 5 days of training followed by a game. Blood was collected from 10 athletes at the start of training (basal), after training (pre-game), and postgame. Their acceleration capacity also was measured pre- and postgame. Blood analysis showed an increase in lactate concentration postgame (13%) and total antioxidant capacity increased both pre-game (13.1%) and postgame (12.7%), all in comparison with basal levels. An oxidative stress marker, protein carbonyl (PC), increased 3-fold over the course of the game, which correlated with a decreased acceleration (r = 0.749). For biomarkers of tissue damage, creatine kinase and aspartate transaminase (AST) increased postgame by 150% and 75%, respectively. The AST variation had a high negative correlation with energy and carbohydrate consumption and a moderate correlation with lipid and vitamin C intake. Protein intake had a positive but moderate correlation with reduced glutathione. The observed correlations suggest that nutritional monitoring can improve exercise physiological homeostasis and that PC serves as a good biomarker for oxidative stress and performance loss.

  10. The Highwire Ubiquitin Ligase Promotes Axonal Degeneration by Tuning Levels of Nmnat Protein

    PubMed Central

    Xiong, Xin; Hao, Yan; Sun, Kan; Li, Jiaxing; Li, Xia; Mishra, Bibhudatta; Soppina, Pushpanjali; Wu, Chunlai; Hume, Richard I.; Collins, Catherine A.

    2012-01-01

    Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury. PMID:23226106

  11. Low Levels of Exhaled Surfactant Protein A Associated With BOS After Lung Transplantation

    PubMed Central

    Ericson, Petrea A.; Mirgorodskaya, Ekaterina; Hammar, Oscar S.; Viklund, Emilia A.; Almstrand, Ann-Charlotte R.; Larsson, Per J-W.; Riise, Gerdt C.; Olin, Anna-Carin

    2016-01-01

    Background There is no clinically available marker for early detection or monitoring of chronic rejection in the form of bronchiolitis obliterans syndrome (BOS), the main long-term complication after lung transplantation. Sampling and analysis of particles in exhaled air is a valid, noninvasive method for monitoring surfactant protein A (SP-A) and albumin in the distal airways. Methods We asked whether differences in composition of exhaled particles can be detected when comparing stable lung transplant recipients (LTRs) (n = 26) with LTRs who develop BOS (n = 7). A comparison between LTRs and a matching group of healthy controls (n = 33) was also conducted. Using a system developed in-house, particles were collected from exhaled air by the principal of inertial impaction before chemical analysis by immunoassays. Results Surfactant protein A in exhaled particles and the SP-A/albumin ratio were lower (P = 0.002 and P = 0.0001 respectively) in the BOS group compared to the BOS-free group. LTRs exhaled higher amount of particles (P < 0.0001) and had lower albumin content (P < 0.0001) than healthy controls. Conclusions We conclude that low levels of SP-A in exhaled particles are associated with increased risk of BOS in LTRs. The possibility that this noninvasive method can be used to predict BOS onset deserves further study with prospective and longitudinal approaches. PMID:27795995

  12. Correlation between erythrocyte sedimentation rate and C-reactive protein level in patients with rheumatic diseases

    PubMed Central

    Kotulska, Anna; Kopeć-Mędrek, Magdalena; Grosicka, Anida; Kubicka, Monika

    2015-01-01

    Objectives Erythrocyte sedimentation rate (ESR) and serum level of C-reactive protein (CRP) are the acute phase reactants most commonly determined in patients with rheumatic diseases. The indices are affected by different factors, but both of them are applied for evaluation of the disease activity in patients with inflammatory disorders of the musculoskeletal system. Material and methods The authors compared the results of ESR and CRP, which were carried out during routine diagnosis in 200 patients admitted to the Department of Rheumatology. Results A significant correlation between ESR and CRP was found (ESR after 1 h/CRP: correlation coefficient 0.6944, ESR after 2 h/CRP: correlation coefficient 0.6126). There was no difference in ESR or CRP between male and female patients, and patients older than 40 years had higher ESR and CRP. Conclusions The obtained results support the usefulness of both indices in the clinical practice of rheumatologists. PMID:27407254

  13. Dietary nutrient levels regulate protein and carbohydrate intake, gluconeogenic/glycolytic flux and blood trehalose level in the insect Manduca sexta L.

    PubMed

    Thompson, S N; Borchardt, D B; Wang, L-W

    2003-03-01

    This study examined the effects of dietary casein and sucrose levels on nutrient intake, and distinguished the effects of carbohydrate and protein consumption on growth, fat content, pyruvate metabolism and blood trehalose level of 5th instar Manduca sexta larvae. Growth increased with increasing casein consumption but was unaffected by carbohydrate intake. Fat content also increased with carbohydrate consumption, but on carbohydrate-free diets fat content increased with increased protein consumption. Blood trehalose level and pyruvate metabolism were examined by nuclear magnetic resonance spectroscopy analysis of blood following administration of (3-(13)C)pyruvate. On diets containing sucrose, blood trehalose increased with increasing carbohydrate intake, and on most diets trehalose was synthesized entirely from dietary sucrose. Pyruvate cycling, indicated by the alanine C2/C3 (13)C enrichment ratio, increased with carbohydrate consumption reflecting increased glycolysis, and pyruvate decarboxylation exceeded carboxylation on all sucrose diets. Larvae that consumed <75 mg/day sucrose were gluconeogenic, based on the [2 (trehalose C6)(Glx C3/C2)]/alanine C2] (13)C enrichment ratio. On carbohydrate-free diets, blood trehalose levels were low and maintained entirely by gluconeogenesis. Blood trehalose level increased with increasing protein intake. Pyruvate cycling was very low, although many insects displayed higher levels of pyruvate decarboxylation than carboxylation. All gluconeogenic larvae displayed alanine (13)C enrichment ratios <0.35 and had blood trehalose levels <50 mM.

  14. Effect of vitamins, protein level and probiotics on immune response of moulted male broiler breeders.

    PubMed

    Khan, R U; Rahman, Z U; Javed, I; Muhammad, F

    2014-08-01

    This study was planned to investigate the comparative effect of vitamins C (L-ascorbic acid), E (DL-α-tocopherol acetate), probiotics, lower than normal protein level (14%) and combination of these treatments on immune response of male broiler breeders after zinc-induced moulting. One hundred and eighty birds at the age of 65 weeks were induced to moult by mixing zinc oxide (ZnO) in feed at the rate 3000 IU/kg of feed. Upon completion of moulting, birds were divided into six groups (five replicates per group) in a completely randomized design and were fed vitamin C (500 IU/kg), vitamin E (100 IU/kg), lower protein level, probiotics (50 mg/l), and a combination of these components, while one group was kept as control. After completion of moulting phase (5 weeks), the treatment effects were tested as in vitro macrophages engulfment percentage, nitric oxide (NO) production, serum antibody titres against Newcastle disease (ND) and infectious bronchitis (IB). The results showed that in vitro macrophage engulfment percentage in unopsonized conditions was significantly higher in vitamin E-supplemented group. In addition, in opsonized condition, the macrophage engulfment percentage was significantly higher in both vitamin E- and C-supplemented groups. The NO (opsonized and unopsonized) production and antibody titre against ND and IB were significantly higher in vitamin E-supplemented group. It was concluded that vitamin E is a better option for enhanced immune response in broiler breeders after zinc-induced moulting.

  15. Comprehensive expression analysis of FSHD candidate genes at the mRNA and protein level.

    PubMed

    Klooster, Rinse; Straasheijm, Kirsten; Shah, Bharati; Sowden, Janet; Frants, Rune; Thornton, Charles; Tawil, Rabi; van der Maarel, Silvère

    2009-12-01

    In facioscapulohumeral muscular dystrophy (FSHD) the majority of patients carry a D4Z4 macrosatellite repeat contraction in the subtelomere of chromosome 4q. Several disease mechanisms have been proposed to explain how repeat contraction causes muscular dystrophy. All proposed mechanisms foresee a change from a closed to a more open chromatin structure followed by loss of control over expression of genes in or proximal to D4Z4. Initially, a distance and residual repeat size-dependent upregulation of the candidate genes FRG2, FRG1 and ANT1 was observed, but most successive expression studies failed to support transcriptional upregulation of 4qter genes. Moreover, chromatin studies do not provide evidence for a cis-spreading mechanism operating at 4qter in FSHD. In part, this inconsistency may be explained by differences in the techniques used, and the use of RNA samples obtained from different muscle groups. The aim of this study is to comprehensively and uniformly study the expression of the FSHD candidate genes FRG1, FRG2, CRYM, ANT1, ALP, PITX1 and LRP2BP at the RNA and protein level in identically processed primary myoblasts, myotubes and quadriceps muscle. Expression was compared between samples obtained from FSHD patients and normal controls with samples from myotonic dystrophy type 1 patients as disease controls. No consistent changes in RNA or protein expression levels were observed between the samples. The one exception was a selective increase in FRG2 mRNA expression in FSHD myotubes. This study provides further evidence that there is no demonstrable consistent, large magnitude, overexpression of any of the FSHD candidate genes.

  16. Endogenous salicylic acid levels correlate with accumulation of pathogenesis-related proteins and virus resistance in tobacco

    SciTech Connect

    Yalpani, N.; Shulaev, V.; Raskin, I. )

    1993-07-01

    Salicylic acid (SA) is hypothesized to be an endogenous regulator of local and systemic disease resistance and an inducer of pathogenesis-related (PR) proteins among plants. High levels of PR proteins have been observed in an uninoculated amphidiploid hybrid of Nicotiana glutinosa [times] N. debneyi, which is highly resistant to tobacco mosaic virus (TMV). Fluoresence, UV, and mass spectral analysis established that the levels of SA in healthy N. glutinosa [times] N. debneyi leaves were 30 times greater than in N. tabacum [open quotes]Xanthi-nc[close quotes] tobacco, which does not constitutively express PR proteins and is less resistant to TMV. Upon TMV-inoculation SA levels increased at least 70-fold leaves of Xanthi-nc but role only slightly in the hybrid. Phloem exudates of N. glutinosa [times] N. debneyi contained at least 500 times more SA than those of Xanthi-nc. SA treatment caused the appearance of PR-1 protein in Xanthi-nc but did not affect constitutively high levels of PR-1 protein in N. glutinosa [times] N. debneyi. In contrast to Xanthi-nc tobacco, TMV-inoculated N. glutinosa [times] N. debneyi kept at 32 C accumulated more than 0.5 [mu]g SA/g fresh weight, maintained high levels of PR proteins, and developed a hypersensitive response to TMV. PR proteins have previously been shown to accumulate in the lower leaves of healthy, flowering Xanthi-nc tobacco, which exhibited increased resistance to TMV. These developmentally induced increases in resistance and PR-1 proteins positively correlated with tissue levels of SA. These results affirm the regulatory role of SA in disease resistance and PR protein production. 31 refs., 9 figs., 1 tab.

  17. The Influence of Tobacco Smoke on Protein and Metal Levels in the Serum of Women during Pregnancy

    PubMed Central

    Wrześniak, Marta; Kepinska, Marta; Królik, Małgorzata; Milnerowicz, Halina

    2016-01-01

    Background Tobacco smoking by pregnant women has a negative effect on fetal development and increases pregnancy risk by changing the oxidative balance and microelements level. Smoking affects the concentration, structure and function of proteins, potentially leading to various negative effects on pregnancy outcomes. Methodology/Principal Findings The influence of tobacco smoke on key protein fractions in smoking and non-smoking healthy pregnant women was determined by capillary electrophoresis (CE). Concentrations of the proteins α1-antitrypsin, α1-acid glycoprotein, α2-macroglobulin and transferrin were determined by ELISA tests. Total protein concentration was measured by the Biuret method. Smoking status was established by cotinine levels. Cadmium (Cd) and Zinc (Zn) concentrations were determined by flame atomic absorption spectrometry and the Zn/Cd ratio was calculated based on these numbers. Smoking women had a 3.7 times higher level of Cd than non-smoking women. Zn levels decreased during pregnancy for all women. The Zn/Cd ratio was three times lower in smoking women. The differences between the changes in the protein profile for smoking and non-smoking women were noted. Regarding proteins, α1-antitrypsin and α2-macroglobulin levels were lower in the non-smoking group than in the smoking group and correlated with Cd levels (r = -0.968, p = 0.032 for non-smokers; r = −0.835, p = 0.019 for smokers). Zn/Cd ratios correlated negatively with α1-, α2- and β-globulins. Conclusions/Significance Exposure to tobacco smoke increases the concentration of Cd in the blood of pregnant women and may lead to an elevated risk of pregnancy disorders. During pregnancy alter concentrations of some proteins. The correlation of Cd with proteins suggests that it is one of the causes of protein aberrations. PMID:27548057

  18. Altered CYP2C9 Activity Following Modulation of CYP3A4 Levels in Human Hepatocytes: an Example of Protein-Protein Interactions

    PubMed Central

    Tweedie, Donald J.; Chan, Tom S.; Tracy, Timothy S.

    2014-01-01

    Cytochrome P450 (P450) protein-protein interactions resulting in modulation of enzyme activities have been well documented using recombinant isoforms. This interaction has been less clearly demonstrated in a more physiologic in vitro system such as human hepatocytes. As an expansion of earlier work (Subramanian et al., 2010), in which recombinant CYP2C9 activity decreased with increasing levels of CYP3A4, the current study modulated CYP3A4 content in human hepatocytes to determine the impact on CYP2C9. Modulation of CYP3A4 levels in situ was enabled by the use of a long-term human hepatocyte culture model (HepatoPac) shown to retain phenotypic hepatocyte function over a number of weeks. The extended period of culture allowed time for knockdown of CYP3A4 protein by small interfering RNA (siRNA) with subsequent recovery, as well as upregulation through induction with a recovery period. CYP3A4 gene silencing resulted in a 60% decrease in CYP3A4 activity and protein levels with a concomitant 74% increase in CYP2C9 activity, with no change in CYP2C9 mRNA levels. Upon removal of siRNA, both CYP2C9 and CYP3A4 activities returned to pre-knockdown levels. Importantly, modulation of CYP3A4 protein levels had no impact on cytochrome P450 reductase activities or levels. However, the possibility for competition for limiting reductase cannot be ruled out. Interestingly, lowering CYP3A4 levels also increased UDP-glucuronosyltransferase 2B7 activity. These studies clearly demonstrate that alterations in CYP3A4 levels can modulate CYP2C9 activity in situ and suggest that further studies are warranted to evaluate the possible clinical consequences of these findings. PMID:25157098

  19. Elevated urinary level of vitamin D-binding protein as a novel biomarker for diabetic nephropathy

    PubMed Central

    TIAN, XIAO-QIN; ZHAO, LI-MIN; GE, JIA-PU; ZHANG, YAN; XU, YAN-CHENG

    2014-01-01

    Improving the early prediction and detection of diabetic nephropathy (DN) remains a great challenge in disease management. The aim of this study was to evaluate the early detection power of urinary vitamin D-binding protein (VDBP) for the diagnosis of DN. Urine samples were obtained from 45 healthy volunteers and 105 diabetic patients with normoalbuminuria (DM group), microalbuminuria (DN1 group) and macroalbuminuria (DN2 group) (n=35 per group). The VDBP expression patterns in urine from patients and controls were quantified by western blot analysis. The excretion levels of urinary VDBP were quantified with enzyme-linked immunosorbent assay. The quantification results were obtained by correcting for creatinine expression and showed that urinary VDBP levels were significantly elevated in the patients of the DN1 and DN2 groups compared with those of the DM group and normal controls (1,011.33±325.30 and 1,406.34±239.66 compared with 466.54±213.63 and 125.48±98.27 ng/mg, respectively) (P<0.001). Receiver operating characteristic analysis of urinary VDBP levels for the diagnosis of DN rendered an optimum cut-off value of 552.243 ng/mg corresponding to 92.86% sensitivity and 85.00% specificity, which also showed an area under the ROC curve of 0.966. In conclusion, the findings of the present study suggest that urinary VDBP may be a potential biomarker for the early detection and prevention of DN. Further studies are required to examine the pathogenic mechanisms of elevated VDBP levels and their role in the diagnosis of DN. PMID:24396416

  20. Diet quality is associated with circulating C-reactive protein but not irisin levels in humans

    PubMed Central

    Park, Kyung Hee; Zaichenko, Lesya; Peter, Patricia; Davis, Cynthia R.; Crowell, Judith A.; Mantzoros, Christos S.

    2015-01-01

    Objective Adherence to a healthy diet has been shown to decrease the incidence of obesity and associated comorbidities. C-reactive protein (CRP) is an established inflammatory marker and irisin was recently identified as a molecule which may play a role in energy regulation and obesity but whether diet alters irisin levels remains unknown. We aimed to investigate the association between circulating irisin, leptin, and CRP levels and dietary quantity and quality using the Alternate Healthy Eating Index (AHEI) and the Alternate Mediterranean Diet Score (aMED). Materials/Methods The study evaluated dietary data and biomarker levels of 151 participants between 2009 and 2011 (71 male vs. 80 female, over 35 years old, obese 43.7%). AHEI and aMED scores were calculated based on data derived from self-administered 110-item food-frequency questionnaires estimating usual nutrient intake over the past year. Cross-sectional associations between dietary quantity, quality, body composition by bioelectric impedance, and biomarker levels including irisin, leptin, and CRP after fasting were assessed. Results CRP, but not irisin, was negatively correlated with AHEI (r = −0.34) and aMED (r = −0.31). Irisin was positively correlated with BMI (r = 0.22), fat mass (r = 0.21), waist circumference (r = 0.24), waist-hip ratio (r = 0.20), leptin (r = 0.32), and CRP (r = 0.25). Participants with the highest AHEI scores tended to have 11.6% lower concentrations of irisin (P for trend =0.09), but they were not significant after adjustment for potential confounders. Better diet quality was associated with lower CRP concentrations (P for trend=0.02) in multivariate model. Percentage of energy from carbohydrate was inversely associated with CRP. Conclusions Unlike CRP, irisin is not associated with dietary quality or quantity. PMID:24315778

  1. Flaxseed oil supplementation decreases C-reactive protein levels in chronic hemodialysis patients.

    PubMed

    Lemos, Joana R N; Alencastro, Mariana Gascue de; Konrath, Anita Vieceli; Cargnin, Marina; Manfro, Roberto Ceratti

    2012-12-01

    Malnutrition and chronic inflammation in dialysis patients negatively impact their survival prognosis, and nutrients, such as omega-3 oils, are postulated to reduce proinflammatory response. In this randomized, double-blind, multicenter, placebo-controlled trial, we investigated the effects of flaxseed oil (FO) on the inflammatory state of patients with chronic renal failure undergoing renal replacement therapy with hemodialysis (HD). We hypothesized that FO supplementation lowers C-reactive protein (CRP) levels. One hundred sixty patients with chronic renal failure who received HD therapy of 3 dialysis units over a 3-month period in South Brazil were included. The patients received blind doses of FO (1 g twice a day) and placebo (mineral oil, 1 g twice a day) for a period of 120 days. Inflammation was observed in 89 patients (61%) at the beginning of the study. There was a correlation between CRP and the body mass index (R(s) = 0.22; P = .022) and high-density lipoprotein cholesterol (R(s) = -0.23; P = .032), and the CRP levels decreased significantly over time in the group that received FO compared with the control group (P < .001). During the study period, 33.3% of the FO group changed from an inflamed to a not-inflamed category, whereas only 16.9% changed in the mineral oil group (P = .04). We conclude that the administration of FO decreases the CRP levels and that inflammation in HD patients appears to be correlated to their body mass index and reduced high-density lipoprotein cholesterol levels. Studies with a larger number of patients and over a longer duration are necessary to corroborate these findings.

  2. Levels of common salivary protein 1 in healthy subjects and periodontal patients

    PubMed Central

    2016-01-01

    Purpose Human saliva, as a vital part of the immune defense system, contains a number of distinct proteins and peptides. Recently human common salivary protein 1 (CSP1) has been identified as an abundant salivary protein and may play a role in promoting the binding of cariogenic bacteria to salivary pellicles. However, nothing else is known regarding the role of CSP1 in periodontology. The aim of this study was to quantify and compare CSP1 levels between healthy subjects and periodontal patients. Methods This controlled clinical study was conducted in periodontally healthy individuals and patients with chronic periodontitis Chonbuk National University Hospital, with Institutional Review Board approval. Whole saliva samples were collected from 36 healthy subjects and 33 chronic periodontitis patients and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immune blotting were conducted to ensure that anti-CSP1 monoclonal antibody (mAb) binds to CSP1 in human saliva. A sandwich enzyme-linked immunosorbent assay (ELISA) system was house-fabricated using mAb-hCSP1#14 and mAb-hCSP1#4 as a capture and a detector mAb, respectively. The CSP1 concentrations in saliva from 36 healthy subjects and 33 periodontal patients were quantified using the CSP1 sandwich ELISA system, and the results were analyzed using the Student’s t-test. Results Immunoblot analysis using mAb-hCSP1 as a probe confirmed that CSP1 in human saliva existed as a single band with a molecular weight of approximately 27-kDa. The quantification of CSP1 concentrations by CSP1 ELISA showed that the median values (25th to 75th percentiles) of periodontal patients and healthy subjects were 9,474 ng/mL (range, 8,434–10,139 ng/mL) and 8,598 ng/mL (range, 7,421–9,877 ng/mL), respectively. The Student’s t-test indicated the presence of a statistically significant difference between the 2 groups (P=0.024). Conclusions The presence of a significant difference in CSP1 levels between healthy

  3. Plasma lactate, GH and GH-binding protein levels in exercise following BCAA supplementation in athletes.

    PubMed

    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 < 0.05 [mean +/- std. err.]). The end-exercise pGH of each athlete was higher than basal (p < 0.05). Furthermore, after the chronic treatment, this end-exercise pGH was higher (but not significantly, p = 0.08) than before this treatment (12.2+/-2.0 ng mL(-1) before vs 33.8+/-13.6 ngmL(-1) after treatment). The end-exercise pGHBP was higher than basal (p < 0.05); and after the 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.

  4. Dormancy alleviation by NO or HCN leading to decline of protein carbonylation levels in apple (Malus domestica Borkh.) embryos.

    PubMed

    Krasuska, Urszula; Ciacka, Katarzyna; Dębska, Karolina; Bogatek, Renata; Gniazdowska, Agnieszka

    2014-08-15

    Deep dormancy of apple (Malus domestica Borkh.) embryos can be overcome by short-term pre-treatment with nitric oxide (NO) or hydrogen cyanide (HCN). Dormancy alleviation of embryos modulated by NO or HCN and the first step of germination depend on temporary increased production of reactive oxygen species (ROS). Direct oxidative attack on some amino acid residues or secondary reactions via reactive carbohydrates and lipids can lead to the formation of protein carbonyl derivatives. Protein carbonylation is a widely accepted covalent and irreversible modification resulting in inhibition or alteration of enzyme/protein activities. It also increases the susceptibility of proteins to proteolytic degradation. The aim of this work was to investigate protein carbonylation in germinating apple embryos, the dormancy of which was removed by pre-treatment with NO or HCN donors. It was performed using a quantitative spectrophotometric method, while patterns of carbonylated protein in embryo axes were analyzed by immunochemical techniques. The highest concentration of protein carbonyl groups was observed in dormant embryos. It declined in germinating embryos pre-treated with NO or HCN, suggesting elevated degradation of modified proteins during seedling formation. A decrease in the concentration of carbonylated proteins was accompanied by modification in proteolytic activity in germinating apple embryos. A strict correlation between the level of protein carbonyl groups and cotyledon growth and greening was detected. Moreover, direct in vitro carbonylation of BSA treated with NO or HCN donors was analyzed, showing action of both signaling molecules as protein oxidation agents.

  5. Gene induction during differentiation of human monocytes into dendritic cells: an integrated study at the RNA and protein levels

    PubMed Central

    Angénieux, Catherine; Fricker, Dominique; Strub, Jean-Marc; Luche, Sylvie; Bausinger, Huguette; Cazenave, Jean-Pierre; Van Dorsselaer, Alain; Hanau, Daniel; de la Salle, Henri; Rabilloud, Thierry

    2001-01-01

    Changes in gene expression occurring during differentiation of human monoytes into dendritic cells were studied at the RNA and protein levels. These studies showed the induction of several gene classes corresponding to various biological functions. These functions encompass of course antigen processing and presentation, cytoskeleton, cell signalling and signal transduction, but also an increase of mitochondrial function and of the protein synthesis machinery, including some, but not all, chaperones. These changes put in perspective the events occurring during this differentiation process. On a more technical point, it appears that the studies carried out at the RNA and protein levels are highly complementary. PMID:11793251

  6. Influence of casein as a percentage of true protein and protein level on color and texture of milks containing 1 and 2% fat.

    PubMed

    Misawa, Noriko; Barbano, David M; Drake, MaryAnne

    2016-07-01

    Combinations of fresh liquid microfiltration retentate of skim milk, ultrafiltered retentate and permeate produced from microfiltration permeate, cream, and dried lactose monohydrate were used to produce a matrix of 20 milks. The milks contained 5 levels of casein as a percentage of true protein of about 5, 25, 50, 75, and 80% and 4 levels of true protein of 3.0, 3.76, 4.34, and 5.0% with constant lactose percentage of 5%. The experiment was replicated twice and repeated for both 1 and 2% fat content. Hunter color measurements, relative viscosity, and fat globule size distribution were measured, and a trained panel documented appearance and texture attributes on all milks. Overall, casein as a percentage of true protein had stronger effects than level of true protein on Hunter L, a, b values, relative viscosity, and fat globule size when using fresh liquid micellar casein concentrates and milk serum protein concentrates produced by a combination of microfiltration and ultrafiltration. As casein as a percentage of true protein increased, the milks became more white (higher L value), less green (lower negative a value), and less yellow (lower b value). Relative viscosity increased and d(0.9) generally decreased with increasing casein as a percentage of true protein. Panelists perceived milks with increasing casein as a percentage of true protein as more white, more opaque, and less yellow. Panelists were able to detect increased throat cling and mouthcoating with increased casein as a percentage of true protein in 2% milks, even when differences in appearance among milks were masked.

  7. Influence of storage and heating on protein glycation levels of processed lactose-free and regular bovine milk products.

    PubMed

    Milkovska-Stamenova, Sanja; Hoffmann, Ralf

    2017-04-15

    Thermal treatment preserves the microbiological safety of milk, but also induces Maillard reactions modifying for example proteins. The purpose of this study was evaluating the influence of consumer behaviors (storage and heating) on protein glycation degrees in bovine milk products. Lactosylation and hexosylation sites were identified in ultra-high temperature (UHT), lactose-free pasteurized, and lactose-free UHT milk (ULF) and infant formula (IF) using tandem mass spectrometry (electron transfer dissociation). Overall, 303 lactosylated and 199 hexosylated peptides were identified corresponding to 170 lactosylation (31 proteins) and 117 hexosylation sites (25 proteins). In quantitative terms, storage increased lactosylation up to fourfold in UHT and IF and hexosylation up to elevenfold in ULF and threefold in IF. These levels increased additionally twofold when the stored samples were heated (40°C). In conclusion, storage and heating appear to influence protein glycation levels in milk at similar or even higher degrees than industrial processing.

  8. Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis.

    PubMed

    Dresios, John; Aschrafi, Armaz; Owens, Geoffrey C; Vanderklish, Peter W; Edelman, Gerald M; Mauro, Vincent P

    2005-02-08

    The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under conditions of mild hypothermia, and Rbm3 has been postulated to facilitate protein synthesis at colder temperatures. To investigate this possibility, Rbm3 was overexpressed as a c-Myc fusion protein in mouse neuroblastoma N2a cells. Cells expressing this fusion protein showed a 3-fold increase in protein synthesis at both 37 degrees C and 32 degrees C compared with control cells. Although polysome profiles of cells expressing the fusion protein and control cells were similar, several differences were noted, suggesting that Rbm3 might enhance the association of 40S and 60S ribosomal subunits at 32 degrees C. Studies to assess a direct interaction of Rbm3 with ribosomes showed that a fraction of Rbm3 was associated with 60S ribosomal subunits in an RNA-independent manner. It appeared unlikely that this association could explain the global enhancement of protein synthesis, however, because cells expressing the Rbm3 fusion protein showed no substantial increase in the size of their monosome and polysome peaks, suggesting that similar numbers of mRNAs were being translated at approximately the same rates. In contrast, a complex that sedimented between the top of the gradient and 40S subunits was less abundant in cells expressing recombinant Rbm3. Further analysis showed that the RNA component of this fraction was microRNA. We discuss the possibility that Rbm3 expression alters global protein synthesis by affecting microRNA levels and suggest that both Rbm3 and microRNAs are part of a homeostatic mechanism that regulates global levels of protein synthesis under normal and cold-stress conditions.

  9. Elevated C-reactive protein levels and metabolic syndrome in the elderly

    PubMed Central

    Zuliani, Giovanni; Volpato, Stefano; Galvani, Matteo; Blè, Alessandro; Bandinelli, Stefania; Corsi, Anna Maria; Lauretani, Fulvio; Maggio, Marcello; Guralnik, Jack M.; Fellin, Renato; Ferrucci, Luigi

    2009-01-01

    Metabolic syndrome (MS) and “low grade” systemic inflammation (LGSI) are very common findings in the older population. Although MS and LGSI have been associated in adults, it is not known what is the real contribution of MS, and its single components, to LGSI in older persons, due to the potential confounding effect of comorbidity and aging. We investigated the relationship between increased C-reactive protein (CRP) plasma levels, a marker of LGSI, and MS in 1044 older (≥65 years) community dwelling Italian individuals enrolled the InChianti study. Metabolic syndrome was defined by the NCEP-ATP III-AHA/NHLBI criteria. High sensitivity CRP (hs.CRP) levels were measured by enzyme-linked immunosorbent assay, and defined as high when >3 mg/L. The overall prevalence of MS was 31%. The prevalence of high hs.CRP was 54.5% in subjects with, and 41.3% in those without MS (p < 0.001). MS was associated with high hs.CRP levels after adjustment for age, gender, and comorbidity (OR: 1.93, 95% CI: 1.46-2.55). Compared to subjects with MS and no LGSI, individuals with MS and LGSI were characterized by higher waist circumference, BMI, and HOMA score. Multivariate logistic regression analysis confirmed the association between waist circumference and high hs.CRP levels in subjects with MS (waist circumference III vs. I tertile OR: 2.60, 95% CI: 1.79-3.77) independent of age, gender, and important confounding variables including comorbidity. Additional analyses, conducted with and without dichotomization of hs.CRP levels, confirmed the central role of waist circumference in the LGSI phenomenon, independent of gender and diagnosis of MS. We conclude that in older individuals, MS is associated with LGSI, but the association is mainly supported by a strong independent correlation between waist circumference and high hs.CRP levels. In the absence of this specific MS component, it seems that the contribution of MS to LGSI would be modest at best. PMID:18845301

  10. Elevated C-reactive protein levels, psychological distress, and depression in 73, 131 individuals.

    PubMed

    Wium-Andersen, Marie Kim; Ørsted, David Dynnes; Nielsen, Sune Fallgaard; Nordestgaard, Børge Grønne

    2013-02-01

    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 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 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, increasing CRP levels were associated with increasing risk for psychological distress and depression (P = 3 × 10-8 to P = 4 × 10-105 for trend). For self-reported use of antidepressants, the odds ratio was 1.38 (95% CI, 1.23-1.55) for CRP levels of 1.01 to 3.00 mg/L, 2.02 (1.77-2.30) for 3.01 to 10.00 mg/L, and 2.70 (2.25-3.25) for greater than 10.00 mg/L compared with 0.01 to 1.00 mg/L. For prescription of antidepressants, the corresponding odds ratios were 1.08 (95% CI, 0.99-1.17), 1.47 (1.33-1.62), and 1.77 (1.52-2.05), respectively; for hospitalization with depression, 1.30 (1.01-1.67), 1.84 (1.39-2.43), and 2.27 (1.54-3.32), respectively. In prospective analyses, increasing CRP levels were also associated with increasing risk for hospitalization with depression (P = 4 × 10-8 for trend). CONCLUSIONS Elevated levels of CRP are associated with increased risk for psychological distress and depression in the general population.

  11. Sleep deprivation and sleep recovery modifies connexin36 and connexin43 protein levels in rat brain.

    PubMed

    Franco-Pérez, Javier; Ballesteros-Zebadúa, Paola; Fernández-Figueroa, Edith A; Ruiz-Olmedo, Isabel; Reyes-Grajeda, Pablo; Paz, Carlos

    2012-01-25

    Gap junctional communication is mainly mediated by connexin36 and connexin43 in neurons and astrocytes, respectively. It has been suggested that connexin36 allows electrical coupling between neurons whereas connexin43 participates in several process including release of ATP. It was recently reported that blockage of gap junctional communication mediated by connexin36 can disrupt the sleep architecture of the rat. However, there is no experimental approach about effects of sleep deprivation on connexins expression. Therefore, we examined in adult male Wistar rats whether protein levels of connexin36 and connexin43 change in pons, hypothalamus, and frontal cortex after 24 h of total sleep deprivation and 4 h of sleep recovery. Western blot revealed that total sleep deprivation significantly decreases the levels of connexin36 in the hypothalamus and this decrease maintains after sleep recovery. Meanwhile, connexin43 is not altered by total sleep deprivation but interestingly the sleep recovery period induces an increase of this connexin. These results suggest that electrical coupling between hypothalamic neurons could be altered by sleep deprivation and that sleep recovery drives changes in connexin43 expression probably as a mechanism related to ATP release and energy regulation during sleep.

  12. Immunological Roles of Elevated Plasma Levels of Matricellular Proteins in Japanese Patients with Pulmonary Tuberculosis

    PubMed Central

    Shiratori, Beata; Zhao, Jingge; Okumura, Masao; Chagan-Yasutan, Haorile; Yanai, Hideki; Mizuno, Kazue; Yoshiyama, Takashi; Idei, Tadashi; Ashino, Yugo; Nakajima, Chie; Suzuki, Yasuhiko; Hattori, Toshio

    2016-01-01

    Elevated matricellular proteins (MCPs), including osteopontin (OPN) and galectin-9 (Gal-9), were observed in the plasma of patients with Manila-type tuberculosis (TB) previously. Here, we quantified plasma OPN, Gal-9, and soluble CD44 (sCD44) by enzyme-linked immunosorbent assay (ELISA), and another 29 cytokines by Luminex assay in 36 patients with pulmonary TB, six subjects with latent tuberculosis (LTBI), and 19 healthy controls (HCs) from Japan for a better understanding of the roles of MCPs in TB. All TB subjects showed positive results of enzyme-linked immunospot assays (ELISPOTs). Spoligotyping showed that 20 out of 36 Mycobacterium tuberculosis (MTB) strains belong to the Beijing type. The levels of OPN, Gal-9, and sCD44 were higher in TB (positivity of 61.1%, 66.7%, and 63.9%, respectively) than in the HCs. Positive correlations between OPN and Gal-9, between OPN and sCD44, and negative correlation between OPN and ESAT-6-ELISPOT response, between chest X-ray severity score of cavitary TB and ESAT-6-ELISPOT response were observed. Instead of OPN, Gal-9, and sCD44, cytokines G-CSF, GM-CSF, IFN-α, IFN-γ, IL-12p70, and IL-1RA levels were higher in Beijing MTB-infected patients. These findings suggest immunoregulatory, rather than inflammatory, effect of MCPs and can advance the understanding of the roles of MCPs in the context of TB pathology. PMID:28025511

  13. Amyloid precursor protein mRNA levels in Alzheimer's disease brain.

    PubMed

    Preece, Paul; Virley, David J; Costandi, Moheb; Coombes, Robert; Moss, Stephen J; Mudge, Anne W; Jazin, Elena; Cairns, Nigel J

    2004-03-17

    Insoluble beta-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene is differentially spliced to produce isoforms that can be classified into those containing a Kunitz-type serine protease inhibitor domain (K(+), APP(751), APP(770), APRP(365) and APRP(563)), and those without (K(-), APP(695) and APP(714)). Given the hypothesis that Abeta is a result of aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors might play an active role in the pathology of AD. We took 513 cerebral cortex samples from 90 AD and 81 control brains and quantified the mRNA isoforms of APP with TaqMan real-time RT-PCR. After adjustment for age at death, brain pH and gender we found a change in the ratio of KPI(+) to KPI(-) mRNA isoforms of APP. Three separate probes, designed to recognise only KPI(+) mRNA species, gave increases of between 28% and 50% in AD brains relative to controls (p=0.002). There was no change in the mRNA levels of KPI-(APP 695) (p=0.898). Therefore, whilst KPI-mRNA levels remained stable the KPI(+) species increased specifically in the AD brains.

  14. Atherogenic diet increases cholesteryl ester transfer protein messenger RNA levels in rabbit liver.

    PubMed

    Quinet, E M; Agellon, L B; Kroon, P A; Marcel, Y L; Lee, Y C; Whitlock, M E; Tall, A R

    1990-02-01

    Cholesteryl ester transfer activity is increased in plasma of cholesterol-fed rabbits. To investigate the mechanisms leading to changes in activity, we measured cholesteryl ester transfer protein (CETP) mass by RIA and CETP mRNA abundance by Northern and slot blot analysis using a human CETP cDNA probe in control (n = 8) and cholesterol-fed rabbits (n = 10). Cholesterol feeding (chow plus 0.5% cholesterol, 10% corn oil) for 30 d increased CETP mass in plasma 3.2-fold in the cholesterol-fed rabbits (12.45 +/- 0.82 micrograms/ml) compared with controls (3.86 +/- 0.38 micrograms/ml). In the hypercholesterolemic rabbit, liver CETP mRNA levels were increased 2.8 times control mRNA levels. Actin, apo E, lecithin-cholesterol acyltransferase, and albumin mRNA abundances were unchanged. In contrast to the widespread tissue distribution in humans, CETP mRNA was not detected in extrahepatic tissues of either control or cholesterol-fed animals. Using a sensitive RNase protection assay, the increase in liver CETP mRNA was detectable within 3 d of beginning the high cholesterol diet. Thus, in response to the atherogenic diet there is an early increase in liver CETP mRNA, probably causing increased CETP synthesis and secretion, and increased plasma CETP. The results indicate that the CETP gene may be regulated by diet-induced changes in lipid metabolism.

  15. Unaltered Glutamate Transporter-1 Protein Levels in Aquaporin-4 Knockout Mice

    PubMed Central

    Hubbard, Jacqueline A.

    2017-01-01

    Maintenance of glutamate and water homeostasis in the brain is crucial to healthy brain activity. Astrocytic glutamate transporter-1 (GLT1) and aquaporin-4 (AQP4) are the main regulators of extracellular glutamate and osmolarity, respectively. Several studies have reported colocalization of GLT1 and AQP4, but the existence of a physical interaction between the two has not been well studied. Therefore, we used coimmunoprecipitation to determine whether a strong interaction exists between these two important molecules in mice on both a CD1 and C57BL/6 background. Furthermore, we used Western blot and immunohistochemistry to examine GLT1 levels in AQP4 knockout (AQP4−/−) mice. An AQP4-GLT1 precipitate was not detected, suggesting the lack of a strong physical interaction between AQP4 and GLT1. In addition, GLT1 protein levels remained unaltered in tissue from CD1 and C57BL/6 AQP4−/− mice. Finally, immunohistochemical analysis revealed that AQP4 and GLT1 do colocalize, but only in a region-specific manner. Taken together, these findings suggest that AQP4 and GLT1 do not have a strong physical interaction between them and are, instead, differentially regulated. PMID:28078912

  16. Protein pheromone expression levels predict and respond to the formation of social dominance networks

    PubMed Central

    Nelson, Adam C.; Cunningham, Christopher B.; Ruff, James S.; Potts, Wayne K.

    2015-01-01

    Communication signals are key regulators of social networks, and are thought to be under selective pressure to honestly reflect social status, including dominance status. The odors of dominants and nondominants differentially influence behavior, and identification of the specific pheromones associated with, and predictive of, dominance status is essential for understanding the mechanisms of network formation and maintenance. In mice, major urinary proteins (MUPs) are excreted in extraordinary large quantities and expression level has been hypothesized to provide an honest signal of dominance status. Here, we evaluate whether MUPs are associated with dominance in wild-derived mice by analyzing expression levels before, during, and after competition for reproductive resources over three days. During competition, dominant males have 24% greater urinary MUP expression than nondominants. The MUP darcin, a pheromone that stimulates female attraction, is predictive of dominance status: dominant males have higher darcin expression before competition. Dominants also have a higher ratio of darcin to other MUPs before and during competition. These differences appear transient, because there are no differences in MUPs or darcin after competition. We also find MUP expression is affected by sire dominance status: socially naive sons of dominant males have lower MUP expression, but this apparent repression is released during competition. A requisite condition for the evolution of communication signals is honesty, and we provide novel insight into pheromones and social networks by showing that MUP and darcin expression is a reliable signal of dominance status, a primary determinant of male fitness in many species. PMID:25867293

  17. Elevated C-reactive protein level predicts lower gastrointestinal tract bleeding

    PubMed Central

    TOMIZAWA, MINORU; SHINOZAKI, FUMINOBU; HASEGAWA, RUMIKO; SHIRAI, YOSHINORI; MOTOYOSHI, YASUFUMI; SUGIYAMA, TAKAO; YAMAMOTO, SHIGENORI; ISHIGE, NAOKI

    2016-01-01

    Lower gastrointestinal (GI) bleeding can be caused by colorectal polyps or cancer. The aim of the present study was to identify blood test variables and medications that can predict lower GI bleeding, which would allow for appropriate colonoscopy. The medical records of patients who underwent colonoscopy from September 2014 to September 2015 were retrospectively analyzed. The selected patients included 278 men (mean age, 67.0±11.5 years) and 249 women (mean age, 69.6±12.0 years). The diagnosis, medications, and blood test variables were compared between patients with and without bleeding. Logistic regression analysis was performed to determine the factors associated with lower GI bleeding. The presence of colorectal polyp and cancer was associated with lower GI bleeding (P=0.0044) with an odds ratio of 6.71 (P=0.0148). No lower GI bleeding was observed in patients taking non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, or anticoagulants. The C-reactive protein (CRP) levels were significantly higher in patients with lower GI bleeding (P=0.0227). The Hb levels were lower in patients with lower GI bleeding, however this finding was not statistically significant (P>0.05). No blood test variable was associated with lower GI bleeding. Elevated CRP was associated with lower GI bleeding, while there was no association between the medications and lower GI bleeding. PMID:27284411

  18. Myostatin protein and RNA transcript levels in adult and developing brook trout.

    PubMed

    Roberts, Steven B; Goetz, Frederick W

    2003-11-28

    Quantitative real-time RT-PCR and Western analysis were used to measure RNA expression of the two brook trout myostatin (MSTN) genes ("ovarian", ov and "brain/muscle", b/m), and levels of MSTN immunoreactive protein (MIP) in developing embryos and muscle of brook trout adults. In developing brook trout embryos, ov and b/m MSTN RNAs and MIP significantly increased 45 days post-fertilization. In adult brook trout, the b/m MSTN form was expressed at higher levels in red versus white muscle regardless of gender or time of year. While few changes were observed in MSTN transcripts in fish sampled throughout the year, a significant increase in the processed 14 kDa MIP was observed at spawning in a tissue specific manner, and differences were observed between males and females. These data, along with promoter sequence analysis of the of b/m and ov genes, support a role for MSTN in muscle growth and development in fish.

  19. Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

    PubMed Central

    Huang, Da Wei; Fanti, Laura; Pak, Daniel T.S.; Botchan, Michael R.; Pimpinelli, Sergio; Kellum, Rebecca

    1998-01-01

    The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae. PMID:9679132

  20. The influences of ambient temperature and crude protein levels on performance and serum biochemical parameters in broilers.

    PubMed

    Liu, Q W; Feng, J H; Chao, Z; Chen, Y; Wei, L M; Wang, F; Sun, R P; Zhang, M H

    2016-04-01

    This study was undertaken to investigate the effects of ambient temperature, crude protein levels and their interaction on performance and serum biochemical parameters of broiler chickens. A total of 216 Arbor Acre broiler chickens (108 males and 108 females) were used in a 2 × 3 factorial arrangement and randomly reared at two temperatures (normal temperature: 23 °C; daily cyclic high temperature: 28-32 °C) and fed on three diets with different crude protein levels (153.3, 183.3 or 213.3 g/kg, with constant essential amino acids) from 28 to 42 days of age. Daily cyclic high ambient temperature decreased final body weight, average daily weight gain, average daily feed intake and serum total protein contents (p < 0.001, p < 0.001, p < 0.001, p = 0.008 respectively), but increased feed/gain, mortality, respiratory rate, rectal temperature, serum uric acid contents and serum creatine kinase activity (p = 0.008, p = 0.003, p < 0.0001, p < 0.0001, p < 0.0001, p = 0.003 respectively), irrespective of crude protein levels. At the ambient temperature, reducing crude protein levels resulted in an increase in feed/gain (p < 0.001), but a decrease in serum total protein and uric acid contents. Only serum creatine kinase activity in broiler chickens was interacted by daily cyclic high ambient temperature and dietary crude protein levels (p = 0.003). These results indicated that daily cyclic high ambient temperature had a great effect on performance and serum biochemical parameters in broiler chickens, whereas dietary crude protein levels affected them partially.

  1. Endothelial cell protein C receptor-mediated redistribution and tissue-level accumulation of factor VIIa

    PubMed Central

    Clark, C A; Vatsyayan, R; Hedner, U; Esmon, C T; Pendurthi, U R; Rao, L V M

    2012-01-01

    Background Recent studies show that activated factor VII (FVIIa) binds to the endothelial cell protein C receptor (EPCR) on the vascular endothelium; however, the importance of this interaction in hemostasis or pathophysiology is unknown. Objective The aim of the present study was to investigate the role of the FVIIa interaction with EPCR on the endothelium in mediating FVIIa transport from the circulation to extravascular tissues. Methods Wild-type, EPCR-deficient or ECPR-over-expressing mice were injected with human recombinant (r)FVIIa (120 μg kg−1 body weight) via the tail vein. At varying time intervals after rFVIIa administration, blood and various tissues were collected to measure FVIIa antigen and activity levels. Tissue sections were analyzed by immunohistochemistry for FVIIa and EPCR. Results The data reveal that, after intravenous (i.v.) injection, rFVIIa rapidly disappears from the blood and associates with the endothelium in an EPCR-dependent manner. Immunohistochemical analyses revealed that the association of FVIIa with the endothelium was maximal at 30 min and thereafter progressively declined. The FVIIa association with the endothelium was undetectable at time points exceeding 24 h post-FVIIa administration. The levels of rFVIIa accumulated in tissue correlate with expression levels of EPCR in mice and FVIIa associated with tissues remained functionally active for periods of at least 7 days. Conclusions The observation that an EPCR-dependent association of FVIIa with the endothelium is most pronounced soon after rFVIIa administration and subsequently declines temporally, combined with the retention of functionally active FVIIa in tissue homogenates for extended periods, indicates that FVIIa binding to EPCR on the endothelium facilitates the transport of FVIIa from circulation to extravascular tissues where TF resides. PMID:22950420

  2. Yeast lacking the amphiphysin family protein Rvs167 is sensitive to disruptions in sphingolipid levels.

    PubMed

    Toume, Moeko; Tani, Motohiro

    2016-08-01

    Rvs167 and Rvs161 in Saccharomyces cerevisiae are amphiphysin family proteins, which are involved in several important cellular events, such as invagination and scission of endocytic vesicles, and actin cytoskeleton organization. It has been reported that cellular dysfunctions caused by deletion of RVS167 or RVS161 are rescued by deletion of specific nonessential sphingolipid-metabolizing enzyme genes. Here, we found that yeast cells lacking RVS167 or RVS161 exhibit a decrease in sphingolipid levels. In rvs167∆ cells, the expression level of Orm2, a negative regulator of serine palmitoyltransferase (SPT) catalyzing the initial step of sphingolipid biosynthesis, was increased in a calcineurin-dependent manner, and the decrease in sphingolipid levels in rvs167∆ cells was reversed on deletion of ORM2. Moreover, repression of both ORM1 and ORM2 expression or overexpression of SPT caused a strong growth defect of rvs167∆ cells, indicating that enhancement of de novo sphingolipid biosynthesis is detrimental to rvs167∆ cells. In contrast, partial repression of LCB1-encoding SPT suppressed abnormal phenotypes caused by the deletion of RVS167, including supersensitivity to high temperature and salt stress, and impairment of endocytosis and actin cytoskeleton organization. In addition, the partial repression of SPT activity suppressed the temperature supersensitivity and abnormal vacuolar morphology caused by deletion of VPS1 encoding a dynamin-like GTPase, which is required for vesicle scission and is functionally closely related to Rvs167/Rvs161, whereas repression of both ORM1 and ORM2 expression in vps1∆ cells caused a growth defect. Thus, it was suggested that proper regulation of SPT activity is indispensable for amphiphysin-deficient cells.

  3. C-Reactive Protein Levels Among U.S. Adults Exposed to Parental Incarceration.

    PubMed

    Boch, Samantha J; Ford, Jodi L

    2015-10-01

    Previous studies have linked childhood adversity to low-grade inflammation via C-reactive protein (CRP) levels. This study analyzed the association between low-grade inflammation and prior biological parental incarceration. Data from the National Longitudinal Study of Adolescent to Adult Health (1994-2008) were analyzed using multinomial logistic regression models. Measures included high-sensitivity (hs)-CRP (<3 mg/L = reference, 3-10 mg/L = low-grade inflammation, and >10 mg/L = acute inflammation), parent incarceration occurring in the child's lifetime, and frequency and timing of incarceration with respect to child's age (0-18 years or >18 years vs. never) of incarceration. Analyses were stratified by child's gender. Final sample sizes were n = 5,396 males and n = 6,447 females for maternal incarceration and n = 4,956 males and n = 5,860 females for paternal incarceration. In models with and without potential mediators, females whose fathers were ever incarcerated were more likely to have hs-CRP levels of 3-10 mg/L than females whose fathers were never incarcerated (adjusted odds ratio [AOR]: 1.44, 95% confidence interval [CI]: [1.09, 1.91]). Additionally, daughter's age (<18 years; AOR: 1.48, 95% CI: [1.11, 1.97]) and frequency of father's incarceration were significant (AOR: 1.24, 95% CI: [1.04, 1.49]). No mediating effects were observed. Males whose fathers were incarcerated when they were ≥18 years were less likely to have hs-CRP levels of 3-10 mg/L than those whose father was never incarcerated; the association was nonsignificant in the mediated model. Further investigation is needed on the physiological effects of exposure to parental incarceration and interventions to support children.

  4. Integrating genomic information with protein sequence and 3D atomic level structure at the RCSB protein data bank.

    PubMed

    Prlić, Andreas; Kalro, Tara; Bhattacharya, Roshni; Christie, Cole; Burley, Stephen K; Rose, Peter W

    2016-12-15

    The Protein Data Bank (PDB) now contains more than 120,000 three-dimensional (3D) structures of biological macromolecules. To allow an interpretation of how PDB data relates to other publicly available annotations, we developed a novel data integration platform that maps 3D structural information across various datasets. This integration bridges from the human genome across protein sequence to 3D structure space. We developed novel software solutions for data management and visualization, while incorporating new libraries for web-based visualization using SVG graphics.

  5. MGMT analysis at DNA, RNA and protein levels in glioblastoma tissue.

    PubMed

    Preusser, Matthias

    2009-04-01

    Evidence from several studies supports that the epigenetic, transcriptional and translational regulation and expression of O6-methylguanine-methyltransferase (MGMT) is relevant for prognostic and predictive considerations in glioblastoma patients. MGMT status is being used as a stratifying factor or eligibility criterion in ongoing and accruing clinical glioblastoma trials. In some cases, there is also interest in MGMT assessment of glioblastoma tissue in the day-to-day clinical setting. However, a number of different methods and protocols have been used for MGMT analysis and it is unclear which methods harbour the greatest potential for translation into routine clinical use. This article reviews methods that have been used for MGMT assessment at DNA-, RNA- and protein-level in glioblastoma with a focus on their potential clinical utility. Conclusions. (1) DNA-based methods for MGMT analysis seem more promising for translation into the clinical setting than RNA- or protein-based methods. However, at present there is lack of data to base recommendations for a specific method or protocol for MGMT testing on. There is a strong need for systematic comparisons and validation of intra- and interlaboratory reproducibility and clinical performance of different methods for MGMT assessment to identify the best method for clinical MGMT testing. (2) The current practice of formalin-fixation of neurosurgical specimens considerably limits the spectrum of methods that can be applied for molecular diagnosis in clinical neuro-oncology. Further studies may be helpful to establish more appropriate protocols for tumour tissue preservation (e.g. identification of alternative fixatives that do not deteriorate DNA and RNA quality).

  6. The effects of dietary protein levels on the population growth, performance, and physiology of honey bee workers during early spring.

    PubMed

    Zheng, Benle; Wu, Zaifu; Xu, Baohua

    2014-01-01

    This study was conducted to investigate the effects of dietary protein levels on honey bee colonies, specifically the population growth, physiology, and longevity of honey bee workers during early spring. Diets containing four different levels of crude protein (25.0, 29.5, 34.0, or 38.5%) and pure pollen (control) were evaluated. Twenty-five colonies of honey bees with sister queens were used in the study. We compared the effects of the different bee diets by measuring population growth, emergent worker weight, midgut proteolytic enzyme activity, hypopharyngeal gland development, and survival. After 48 d, the cumulative number of workers produced by the colonies ranged from 22,420 to 29,519, providing a significant fit to a quadratic equation that predicts the maximum population growth when the diet contains 31.7% crude protein. Significantly greater emergent worker weight, midgut proteolytic enzyme activity, hypopharyngeal gland acini, and survival were observed in the colonies that were fed diets containing 34.0% crude protein compared with the other crude protein levels. Although higher emergent worker weight and survival were observed in the colonies that were fed the control diet, there were no significant differences between the control colonies and the colonies that were fed 34.0% crude protein. Based on these results, we concluded that a dietary crude protein content of 29.5-34.0% is recommended to maximize the reproduction rate of honey bee colonies in early spring.

  7. Leveling

    USGS Publications Warehouse

    1966-01-01

    Geodetic leveling by the U.S. Geological Survey provides a framework of accurate elevations for topographic mapping. Elevations are referred to the Sea Level Datum of 1929. Lines of leveling may be run either with automatic or with precise spirit levels, by either the center-wire or the three-wire method. For future use, the surveys are monumented with bench marks, using standard metal tablets or other marking devices. The elevations are adjusted by least squares or other suitable method and are published in lists of control.

  8. Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

    PubMed

    Geary, Bethany; Magee, Kieran; Cash, Phillip; Young, Iain S; Whitfield, Phillip D; Doherty, Mary K

    2016-05-01

    The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates.

  9. Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover

    NASA Technical Reports Server (NTRS)

    Mooney, D. J.; Hansen, L. K.; Langer, R.; Vacanti, J. P.; Ingber, D. E.

    1994-01-01

    Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and

  10. Maternal protein restriction induced-hypertension is associated to oxidative disruption at transcriptional and functional levels in the medulla oblongata.

    PubMed

    de Brito Alves, José L; de Oliveira, Jéssica M D; Ferreira, Diorginis J S; Barros, Monique A de V; Nogueira, Viviane O; Alves, Débora S; Vidal, Hubert; Leandro, Carol G; Lagranha, Cláudia J; Pirola, Luciano; da Costa-Silva, João H

    2016-12-01

    Maternal protein restriction during pregnancy and lactation predisposes the adult offspring to sympathetic overactivity and arterial hypertension. Although the underlying mechanisms are poorly understood, dysregulation of the oxidative balance has been proposed as a putative trigger of neural-induced hypertension. The aim of the study was to evaluate the association between the oxidative status at transcriptional and functional levels in the medulla oblongata and maternal protein restriction induced-hypertension. Wistar rat dams were fed a control (normal protein; 17% protein) or a low protein ((Lp); 8% protein) diet during pregnancy and lactation, and male offspring was studied at 90 days of age. Direct measurements of baseline arterial blood pressure (ABP) and heart rate (HR) were recorded in awakened offspring. In addition, quantitative RT-PCR was used to assess the mRNA expression of superoxide dismutase 1 (SOD1) and 2 (SOD2), catalase (CAT), glutathione peroxidase (GPx), Glutamatergic receptors (Grin1, Gria1 and Grm1) and GABA(A)-receptor-associated protein like 1 (Gabarapl1). Malondialdehyde (MDA) levels, CAT and SOD activities were examined in ventral and dorsal medulla. Lp rats exhibited higher ABP. The mRNA expression levels of SOD2, GPx and Gabarapl1 were down regulated in medullary tissue of Lp rats (P<.05, t test). In addition, we observed that higher MDA levels were associated to decreased SOD (approximately 45%) and CAT (approximately 50%) activities in ventral medulla. Taken together, our data suggest that maternal protein restriction induced-hypertension is associated with medullary oxidative dysfunction at transcriptional level and with impaired antioxidant capacity in the ventral medulla.

  11. Regional protein levels of cytosolic phospholipase A2 and cyclooxygenase-2 in Rhesus monkey brain as a function of age

    PubMed Central

    Weerasinghe, Gayani R.; Coon, Steven L.; Bhattacharjee, Abesh Kumar; Harry, G. Jean; Bosetti, Francesca

    2006-01-01

    Limited evidence suggests that brain cytosolic phospholipase A2 (cPLA2), which selectively releases arachidonic acid (AA) from membrane phospholipids, and cyclooxygenase-2 (COX-2), the rate-limiting enzyme for AA metabolism to prostanoids, change as a function of normal aging. In this study, we examined the protein levels of cPLA2 and COX-2 enzymes in hippocampus, frontal pole and cerebellum from young (2–5 year-old), middle-aged (8–11 year-old) and old (23 year-old) male and female Rhesus monkeys. In the cerebellum, cPLA2 protein level was higher in the young brain as compared to levels seen at both middle-aged and old. Similarly, in the frontal pole, the young brain showed a higher level of COX-2 protein as compared to the levels seen at both older ages. For both, once an animal reached 8–11 years of age the levels appeared to remain relatively constant over the next decade. Immunohistochemistry of COX-2 protein within the brain demonstrated no significant change in the localization to neurons within the frontal pole. In the young brain, the distribution of a low level of COX-2 protein within numerous neurons was different than the decreased number of neurons stained at a greater intensity in the adult brain. Based on the previous reports of localization of cPLA2 and COX-2 at post-synaptic sites in neurons results from the current study suggest that the elevated protein levels of the two enzymes seen in the younger brain is related to the greater potential for synaptic plasticity across multiple neurons as a function of age and that cPLA2 and COX-2 may be considered as post-synaptic markers. PMID:16716827

  12. Identification of novel candidate drivers connecting different dysfunctional levels for lung adenocarcinoma using protein-protein interactions and a shortest path approach

    PubMed Central

    Chen, Lei; Huang, Tao; Zhang, Yu-Hang; Jiang, Yang; Zheng, Mingyue; Cai, Yu-Dong

    2016-01-01

    Tumors are formed by the abnormal proliferation of somatic cells with disordered growth regulation under the influence of tumorigenic factors. Recently, the theory of “cancer drivers” connects tumor initiation with several specific mutations in the so-called cancer driver genes. According to the differentiation of four basic levels between tumor and adjacent normal tissues, the cancer drivers can be divided into the following: (1) Methylation level, (2) microRNA level, (3) mutation level, and (4) mRNA level. In this study, a computational method is proposed to identify novel lung adenocarcinoma drivers based on dysfunctional genes on the methylation, microRNA, mutation and mRNA levels. First, a large network was constructed using protein-protein interactions. Next, we searched all of the shortest paths connecting dysfunctional genes on different levels and extracted new candidate genes lying on these paths. Finally, the obtained candidate genes were filtered by a permutation test and an additional strict selection procedure involving a betweenness ratio and an interaction score. Several candidate genes remained, which are deemed to be related to two different levels of cancer. The analyses confirmed our assertions that some have the potential to contribute to the tumorigenesis process on multiple levels. PMID:27412431

  13. Identification of novel candidate drivers connecting different dysfunctional levels for lung adenocarcinoma using protein-protein interactions and a shortest path approach

    NASA Astrophysics Data System (ADS)

    Chen, Lei; Huang, Tao; Zhang, Yu-Hang; Jiang, Yang; Zheng, Mingyue; Cai, Yu-Dong

    2016-07-01

    Tumors are formed by the abnormal proliferation of somatic cells with disordered growth regulation under the influence of tumorigenic factors. Recently, the theory of “cancer drivers” connects tumor initiation with several specific mutations in the so-called cancer driver genes. According to the differentiation of four basic levels between tumor and adjacent normal tissues, the cancer drivers can be divided into the following: (1) Methylation level, (2) microRNA level, (3) mutation level, and (4) mRNA level. In this study, a computational method is proposed to identify novel lung adenocarcinoma drivers based on dysfunctional genes on the methylation, microRNA, mutation and mRNA levels. First, a large network was constructed using protein-protein interactions. Next, we searched all of the shortest paths connecting dysfunctional genes on different levels and extracted new candidate genes lying on these paths. Finally, the obtained candidate genes were filtered by a permutation test and an additional strict selection procedure involving a betweenness ratio and an interaction score. Several candidate genes remained, which are deemed to be related to two different levels of cancer. The analyses confirmed our assertions that some have the potential to contribute to the tumorigenesis process on multiple levels.

  14. Copper uptake is required for pyrrolidine dithiocarbamate-mediated oxidation and protein level increase of p53 in cells.

    PubMed Central

    Furuta, Saori; Ortiz, Fausto; Zhu Sun, Xiu; Wu, Hsiao-Huei; Mason, Andrew; Momand, Jamil

    2002-01-01

    The p53 tumour-suppressor protein is a transcription factor that activates the expression of genes involved in cell cycle arrest, apoptosis and DNA repair. The p53 protein is vulnerable to oxidation at cysteine thiol groups. The metal-chelating dithiocarbamates, pyrrolidine dithiocarbamate (PDTC), diethyldithiocarbamate, ethylene(bis)dithiocarbamate and H(2)O(2) were tested for their oxidative effects on p53 in cultured human breast cancer cells. Only PDTC oxidized p53, although all oxidants tested increased the p53 level. Inductively coupled plasma MS analysis indicated that the addition of 60 microM PDTC increased the cellular copper concentration by 4-fold, which was the highest level of copper accumulated amongst all the oxidants tested. Bathocuproinedisulphonic acid, a membrane-impermeable Cu(I) chelator inhibited the PDTC-mediated copper accumulation. Bathocuproinedisulphonic acid as well as the hydroxyl radical scavenger d-mannitol inhibited the PDTC-dependent increase in p53 protein and oxidation. Our results show that a low level of copper accumulation in the range of 25-40 microg/g of cellular protein increases the steady-state levels of p53. At copper accumulation levels higher than 60 microg/g of cellular protein, p53 is oxidized. These results suggest that p53 is vulnerable to free radical-mediated oxidation at cysteine residues. PMID:11964141

  15. Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

    NASA Technical Reports Server (NTRS)

    Evans, G. L.; Morey-Holton, E.; Turner, R. T.

    1998-01-01

    In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-alpha(1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

  16. E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors

    PubMed Central

    Zhang, Yan; Yokoyama, Shigetoshi; Herriges, John C.; Zhang, Zhen; Young, Randee E.; Verheyden, Jamie M.; Sun, Xin

    2016-01-01

    The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis. PMID:27335464

  17. Effects of balanced dietary protein levels on egg production and egg quality parameters of individual commercial layers.

    PubMed

    Shim, M Y; Song, E; Billard, L; Aggrey, S E; Pesti, G M; Sodsee, P

    2013-10-01

    The effects of a series of balanced dietary protein levels on egg production and egg quality parameters of laying hens from 18 through 74 wk of age were investigated. One hundred forty-four pullets (Bovans) were randomly assigned to individual cages with separate feeders including 3 different protein level series of isocaloric diets. Diets were separated into 4 phases of 18-22, 23-32, 33-44, and 45-74 wk of age. The high protein (H) series contained 21.62, 19.05, 16.32, and 16.05% CP, respectively. Medium protein (M) and low protein (L) series were 2 and 4% lower in balanced dietary protein. The results clearly demonstrated that the balanced dietary protein level was a limiting factor for BW, ADFI, egg weight, hen day egg production (HDEP), and feed per kilogram of eggs. Feeding with the L series resulted in lower ADFI and HDEP (90.33% peak production) and more feed per kilogram of eggs compared with the H or M series (HDEP; 93.23 and 95.68% peak production, monthly basis). Egg weight responded in a linear manner to balanced dietary protein level (58.78, 55.94, and 52.73 g for H, M, and L, respectively). Feed intake of all hens, but especially those in the L series, increased considerably after wk 54 when the temperature of the house decreased due to winter conditions. Thus, hens fed the L series seemed particularly dependent on house temperature to maintain BW, ADFI, and HDEP. For egg quality parameters, percent yolk, Haugh units, and egg specific gravity were similar regardless of diets. Haugh units were found to be greatly affected by the variation of housing temperature (P = 0.025). Maximum performance cannot always be expected to lead to maximum profits. Contrary to the idea of a daily amino acid requirement for maximum performance, these results may be used to determine profit-maximizing levels of balanced dietary protein based on the cost of protein and returns from different possible protein levels that may be fed.

  18. Atomic-level description of protein-lipid interactions using an accelerated membrane model.

    PubMed

    Baylon, Javier L; Vermaas, Josh V; Muller, Melanie P; Arcario, Mark J; Pogorelov, Taras V; Tajkhorshid, Emad

    2016-07-01

    Peripheral membrane proteins are structurally diverse proteins that are involved in fundamental cellular processes. Their activity of these proteins is frequently modulated through their interaction with cellular membranes, and as a result techniques to study the interfacial interaction between peripheral proteins and the membrane are in high demand. Due to the fluid nature of the membrane and the reversibility of protein-membrane interactions, the experimental study of these systems remains a challenging task. Molecular dynamics simulations offer a suitable approach to study protein-lipid interactions; however, the slow dynamics of the lipids often prevents sufficient sampling of specific membrane-protein interactions in atomistic simulations. To increase lipid dynamics while preserving the atomistic detail of protein-lipid interactions, in the highly mobile membrane-mimetic (HMMM) model the membrane core is replaced by an organic solvent, while short-tailed lipids provide a nearly complete representation of natural lipids at the organic solvent/water interface. Here, we present a brief introduction and a summary of recent applications of the HMMM to study different membrane proteins, complementing the experimental characterization of the presented systems, and we offer a perspective of future applications of the HMMM to study other classes of membrane proteins. This article is part of a Special Issue entitled: Membrane proteins edited by J.C. Gumbart and Sergei Noskov.

  19. Computational Design of Self-Assembling Protein Nanomaterials with Atomic Level Accuracy

    SciTech Connect

    King, Neil P.; Sheffler, William; Sawaya, Michael R.; Vollmar, Breanna S.; Sumida, John P.; André, Ingemar; Gonen, Tamir; Yeates, Todd O.; Baker, David

    2015-09-17

    We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.

  20. In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

    PubMed Central

    Butko, Margaret T.; Savas, Jeffrey N.; Friedman, Beth; Delahunty, Claire; Ebner, Ford; Yates, John R.; Tsien, Roger Y.

    2013-01-01

    Postnatal bilateral whisker trimming was used as a model system to test how synaptic proteomes are altered in barrel cortex by sensory deprivation during synaptogenesis. Using quantitative mass spectrometry, we quantified more than 7,000 synaptic proteins and identified 89 significantly reduced and 161 significantly elevated proteins in sensory-deprived synapses, 22 of which were validated by immunoblotting. More than 95% of quantified proteins, including abundant synaptic proteins such as PSD-95 and gephyrin, exhibited no significant difference under high- and low-activity rearing conditions, suggesting no tissue-wide changes in excitatory or inhibitory synaptic density. In contrast, several proteins that promote mature spine morphology and synaptic strength, such as excitatory glutamate receptors and known accessory factors, were reduced significantly in deprived synapses. Immunohistochemistry revealed that the reduction in SynGAP1, a postsynaptic scaffolding protein, was restricted largely to layer I of barrel cortex in sensory-deprived rats. In addition, protein-degradation machinery such as proteasome subunits, E2 ligases, and E3 ligases, accumulated significantly in deprived synapses, suggesting targeted synaptic protein degradation under sensory deprivation. Importantly, this screen identified synaptic proteins whose levels were affected by sensory deprivation but whose synaptic roles have not yet been characterized in mammalian neurons. These data demonstrate the feasibility of defining synaptic proteomes under different sensory rearing conditions and could be applied to elucidate further molecular mechanisms of sensory development. PMID:23382246

  1. In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation.

    PubMed

    Butko, Margaret T; Savas, Jeffrey N; Friedman, Beth; Delahunty, Claire; Ebner, Ford; Yates, John R; Tsien, Roger Y

    2013-02-19

    Postnatal bilateral whisker trimming was used as a model system to test how synaptic proteomes are altered in barrel cortex by sensory deprivation during synaptogenesis. Using quantitative mass spectrometry, we quantified more than 7,000 synaptic proteins and identified 89 significantly reduced and 161 significantly elevated proteins in sensory-deprived synapses, 22 of which were validated by immunoblotting. More than 95% of quantified proteins, including abundant synaptic proteins such as PSD-95 and gephyrin, exhibited no significant difference under high- and low-activity rearing conditions, suggesting no tissue-wide changes in excitatory or inhibitory synaptic density. In contrast, several proteins that promote mature spine morphology and synaptic strength, such as excitatory glutamate receptors and known accessory factors, were reduced significantly in deprived synapses. Immunohistochemistry revealed that the reduction in SynGAP1, a postsynaptic scaffolding protein, was restricted largely to layer I of barrel cortex in sensory-deprived rats. In addition, protein-degradation machinery such as proteasome subunits, E2 ligases, and E3 ligases, accumulated significantly in deprived synapses, suggesting targeted synaptic protein degradation under sensory deprivation. Importantly, this screen identified synaptic proteins whose levels were affected by sensory deprivation but whose synaptic roles have not yet been characterized in mammalian neurons. These data demonstrate the feasibility of defining synaptic proteomes under different sensory rearing conditions and could be applied to elucidate further molecular mechanisms of sensory development.

  2. Effect of experimental design and protein substitution strategy on the outcome of feeding different levels of protein in primiparous dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inferences about lactation responses to diet have been hypothesized to be affected by the use of change-over instead of continuous experimental designs. A direct test of this hypothesis has not been well studied. Additionally, when dietary protein level is changed it must occur through dilution with...

  3. Unravelling Protein-DNA Interactions at Molecular Level: A DFT and NCI Study.

    PubMed

    González, J; Baños, I; León, I; Contreras-García, J; Cocinero, E J; Lesarri, A; Fernández, J A; Millán, J

    2016-02-09

    Histone-DNA interactions were probed computationally at a molecular level, by characterizing the bimolecular clusters constituted by selected amino acid derivatives with polar (asparagine and glutamine), nonpolar (alanine, valine, and isoleucine), and charged (arginine) side chains and methylated pyrimidinic (1-methylcytosine and 1-methylthymine) and puric (9-methyladenine and 9-methylguanine) DNA bases. The computational approach combined different methodologies: a molecular mechanics (MMFFs forced field) conformational search and structural and vibrational density-functional calculations (M06-2X with double and triple-ζ Pople's basis sets). To dissect the interactions, intermolecular forces were analyzed with the Non-Covalent Interactions (NCI) analysis. The results for the 24 different clusters studied show a noticeable correlation between the calculated binding energies and the propensities for protein-DNA base interactions found in the literature. Such correlation holded even for the interaction of the selected amino acid derivatives with Watson and Crick pairs. Therefore, the balance between hydrogen bonds and van der Waals interactions (specially stacking) in the control of the final shape of the investigated amino acid-DNA base pairs seems to be well reproduced in dispersion-corrected DFT molecular models, reinforcing the idea that the specificity between the amino acids and the DNA bases play an important role in the regulation of DNA.

  4. JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation

    PubMed Central

    Besancenot, Rodolphe; Roos-Weil, Damien; Tonetti, Carole; Abdelouahab, Hadjer; Lacout, Catherine; Pasquier, Florence; Willekens, Christophe; Rameau, Philippe; Lecluse, Yann; Micol, Jean-Baptiste; Constantinescu, Stefan N.; Vainchenker, William; Solary, Eric

    2014-01-01

    Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses. PMID:25143485

  5. DNA-dependent homodimerization, sub-cellular partitioning, and protein destabilization control WUSCHEL levels and spatial patterning.

    PubMed

    Rodriguez, Kevin; Perales, Mariano; Snipes, Stephen; Yadav, Ram Kishor; Diaz-Mendoza, Mercedes; Reddy, G Venugopala

    2016-10-11

    The homeodomain transcription factor WUSCHEL (WUS) promotes stem cell maintenance in inflorescence meristems of Arabidopsis thaliana WUS, which is synthesized in the rib meristem, migrates and accumulates at lower levels in adjacent cells. Maintenance of WUS protein levels and spatial patterning distribution is not well-understood. Here, we show that the last 63-aa stretch of WUS is necessary for maintaining different levels of WUS protein in the rib meristem and adjacent cells. The 63-aa region contains the following transcriptional regulatory domains: the acidic region, the WUS-box, which is conserved in WUS-related HOMEOBOX family members, and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR-like) domain. Our analysis reveals that the opposing functions of WUS-box, which is required for nuclear retention, and EAR-like domain, which participates in nuclear export, are necessary to maintain higher nuclear levels of WUS in cells of the rib meristem and lower nuclear levels in adjacent cells. We also show that the N-terminal DNA binding domain, which is required for both DNA binding and homodimerization, along with the homodimerization sequence located in the central part of the protein, restricts WUS from spreading excessively and show that the homodimerization is critical for WUS function. Our analysis also reveals that a higher level of WUS outside the rib meristem leads to protein destabilization, suggesting a new tier of regulation in WUS protein regulation. Taken together our data show that processes that influence WUS protein levels and spatial distribution are highly coupled to its transcriptional activity.

  6. Integrating genomic information with protein sequence and 3D atomic level structure at the RCSB protein data bank

    PubMed Central

    Prlić, Andreas; Kalro, Tara; Bhattacharya, Roshni; Christie, Cole; Burley, Stephen K.; Rose, Peter W.

    2016-01-01

    Summary: The Protein Data Bank (PDB) now contains more than 120,000 three-dimensional (3D) structures of biological macromolecules. To allow an interpretation of how PDB data relates to other publicly available annotations, we developed a novel data integration platform that maps 3D structural information across various datasets. This integration bridges from the human genome across protein sequence to 3D structure space. We developed novel software solutions for data management and visualization, while incorporating new libraries for web-based visualization using SVG graphics. Availability and Implementation: The new views are available from http://www.rcsb.org and software is available from https://github.com/rcsb/. Contact: andreas.prlic@rcsb.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27551105

  7. Use of estimated evolutionary strength at the codon level improves the prediction of disease-related protein mutations in humans.

    PubMed

    Capriotti, Emidio; Arbiza, Leonardo; Casadio, Rita; Dopazo, Joaquín; Dopazo, Hernán; Marti-Renom, Marc A

    2008-01-01

    Predicting the functional impact of protein variation is one of the most challenging problems in bioinformatics. A rapidly growing number of genome-scale studies provide large amounts of experimental data, allowing the application of rigorous statistical approaches for predicting whether a given single point mutation has an impact on human health. Up until now, existing methods have limited their source data to either protein or gene information. Novel in this work, we take advantage of both and focus on protein evolutionary information by using estimated selective pressures at the codon level. Here we introduce a new method (SeqProfCod) to predict the likelihood that a given protein variant is associated with human disease or not. Our method relies on a support vector machine (SVM) classifier trained using three sources of information: protein sequence, multiple protein sequence alignments, and the estimation of selective pressure at the codon level. SeqProfCod has been benchmarked with a large dataset of 8,987 single point mutations from 1,434 human proteins from SWISS-PROT. It achieves 82% overall accuracy and a correlation coefficient of 0.59, indicating that the estimation of the selective pressure helps in predicting the functional impact of single-point mutations. Moreover, this study demonstrates the synergic effect of combining two sources of information for predicting the functional effects of protein variants: protein sequence/profile-based information and the evolutionary estimation of the selective pressures at the codon level. The results of large-scale application of SeqProfCod over all annotated point mutations in SWISS-PROT (available for download at http://sgu.bioinfo.cipf.es/services/Omidios/; last accessed: 24 August 2007), could be used to support clinical studies.

  8. SET overexpression in HEK293 cells regulates mitochondrial uncoupling proteins levels within a mitochondrial fission/reduced autophagic flux scenario

    SciTech Connect

    Almeida, Luciana O.; Goto, Renata N.; Neto, Marinaldo P.C.; Sousa, Lucas O.; Curti, Carlos; Leopoldino, Andréia M.

    2015-03-06

    We hypothesized that SET, a protein accumulated in some cancer types and Alzheimer disease, is involved in cell death through mitochondrial mechanisms. We addressed the mRNA and protein levels of the mitochondrial uncoupling proteins UCP1, UCP2 and UCP3 (S and L isoforms) by quantitative real-time PCR and immunofluorescence as well as other mitochondrial involvements, in HEK293 cells overexpressing the SET protein (HEK293/SET), either in the presence or absence of oxidative stress induced by the pro-oxidant t-butyl hydroperoxide (t-BHP). SET overexpression in HEK293 cells decreased UCP1 and increased UCP2 and UCP3 (S/L) mRNA and protein levels, whilst also preventing lipid peroxidation and decreasing the content of cellular ATP. SET overexpression also (i) decreased the area of mitochondria and increased the number of organelles and lysosomes, (ii) increased mitochondrial fission, as demonstrated by increased FIS1 mRNA and FIS-1 protein levels, an apparent accumulation of DRP-1 protein, and an increase in the VDAC protein level, and (iii) reduced autophagic flux, as demonstrated by a decrease in LC3B lipidation (LC3B-II) in the presence of chloroquine. Therefore, SET overexpression in HEK293 cells promotes mitochondrial fission and reduces autophagic flux in apparent association with up-regulation of UCP2 and UCP3; this implies a potential involvement in cellular processes that are deregulated such as in Alzheimer's disease and cancer. - Highlights: • SET, UCPs and autophagy prevention are correlated. • SET action has mitochondrial involvement. • UCP2/3 may reduce ROS and prevent autophagy. • SET protects cell from ROS via UCP2/3.

  9. Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level, Genotype and Developmental Stages

    PubMed Central

    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 (P<0.05) gradually with increasing age. Bama mini-pigs had generally higher (P<0.05) muscle concentrations of flavor-related 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

  10. Effect of dietary crude protein level and degradability on ruminal fermentation and nitrogen utilization in lactating dairy cows.

    PubMed

    Hristov, A N; Etter, R P; Ropp, J K; Grandeen, K L

    2004-11-01

    The objectives of this experiment were to investigate the effects of two ruminally degradable protein (RDP) levels in diets containing similar ruminally undegradable protein (RUP) and metabolizable protein (MP) concentrations on ruminal fermentation, digestibility, and transfer of ruminal ammonia N into milk protein in dairy cows. Four ruminally and duodenally cannulated Holstein cows were allocated to two dietary treatments in a crossover design. The diets (adequate RDP [ARDP] and high RDP [HRDP]), had similar concentrations of RUP and MP, but differed in CP/RDP content. Ruminal ammonia was labeled with 15N and secretion of tracer in milk protein was determined for a period of 120 h. Ammonia concentration in the rumen tended to be greater (P = 0.06) with HRDP than with ARDP. Microbial N flow to the duodenum, ruminal digestibility of dietary nutrients, DMI, milk yield, fat content, and protein content and yield were not statistically different between diets. There was a tendency (P = 0.07) for increased urinary N excretion, and blood plasma and milk urea N concentrations were greater (P = 0.002 and P = 0.01, respectively) with HRDP compared with ARDP. Milk N efficiency was decreased (P = 0.01) by the HRDP diet. The cumulative secretion of ammonia 15N into milk protein, as a proportion of 15N dosed intraruminally, was greater (P = 0.003) with ARDP than with HRDP. The proportions of bacterial protein originating from ammonia N and milk protein originating from bacterial or ammonia N averaged 43, 61, and 26% and were not affected by diet. This experiment indicated that excess RDP in the diet of lactating dairy cows could not be efficiently utilized for microbial protein synthesis and was largely lost through urinary N excretion. At a similar MP supply, increased CP or RDP concentration of the diet would result in decreased efficiency of conversion of dietary N into milk protein and less efficient use of ruminal ammonia N for milk protein syntheses.

  11. The Relationship between Environmental Dioxygen and Iron-Sulfur Proteins Explored at the Genome Level

    PubMed Central

    Andreini, Claudia; Rosato, Antonio; Banci, Lucia

    2017-01-01

    About 2 billion years ago, the atmosphere of the Earth experienced a great change due to the buildup of dioxygen produced by photosynthetic organisms. This transition caused a reduction of iron bioavailability and at the same time exposed living organisms to the threat of oxidative stress. Iron-sulfur (Fe-S) clusters require iron ions for their biosynthesis and are labile if exposed to reactive oxygen species. To assess how the above transition influenced the usage of Fe-S clusters by organisms, we compared the distribution of the Fe-S proteins encoded by the genomes of more than 400 prokaryotic organisms as a function of their dioxygen requirements. Aerobic organisms use less Fe-S proteins than the majority of anaerobic organisms with a similar genome size. Furthermore, aerobes have evolved specific Fe-S proteins that bind the less iron-demanding and more chemically stable Fe2S2 clusters while reducing the number of Fe4S4-binding proteins in their genomes. However, there is a shared core of Fe-S protein families composed mainly by Fe4S4-binding proteins. Members of these families are present also in humans. The distribution of human Fe-S proteins within cell compartments shows that mitochondrial proteins are inherited from prokaryotic proteins of aerobes, whereas nuclear and cytoplasmic Fe-S proteins are inherited from anaerobic organisms. PMID:28135316

  12. Relationship between VEGF Gene Polymorphisms and Serum VEGF Protein Levels in Patients with Rheumatoid Arthritis

    PubMed Central

    Paradowska-Gorycka, Agnieszka; Pawlik, Andrzej; Romanowska-Prochnicka, Katarzyna; Haladyj, Ewa; Malinowski, Damian; Stypinska, Barbara; Manczak, Malgorzata; Olesinska, Marzena

    2016-01-01

    lower than the number of women with wild type allele -2578A (p = 0.006). Serum VEGF levels were significantly higher in RA patients than in control groups (both p = 0,0001). Conclusion Present findings indicated that VEGF genetic polymorphism as well as VEGF protein levels may be associated with the susceptibility to RA in the Polish population. PMID:27513931

  13. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    SciTech Connect

    Chen, Yi; Pirisi, Lucia; Creek, Kim E.

    2013-09-15

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.

  14. Effects of copper on chlorophyll, proline, protein and abscisic acid level of sunflower (Helianthus annuus L.) seedlings.

    PubMed

    Zengin, Fikriye Kirbag; Kirbag, Sevda

    2007-07-01

    The effect of copperchloride (CuCl2) on the level of chlorophyll (a+b), proline, protein and abscisic acid in sunflower (Helianthus annuus L.) seedlings were investigated Control and copper treated (0.4, 0.5 and 0.6 mM) seedlings were grown for ten days in Hoagland solution. Abscisic acid content was determined in root, shoot and leaf tissues of seedlings by HPLC. Copper stress caused significant increase of the abscisic acid contents in roots, shoots and leaves of seedlings. The increase was dependent on the copper salt concentration. Enhanced accumulation of proline in the leaves of seedlings exposed to copper was determined, as well as a decrease of chlorophyll (a+b) and total protein (p < 0.05 or p < 0.01). It was observed that the level of chlorophyll (a+b) and total protein (p < 0.05 or p < 0.01) remarkably decreased as copper concentration increased to 0.6 mM, although the levels of proline and abscisic acid in the leaves of plants were increased--a dose-depended behavior The same trends were also observed with the level of abscisic acid of stems and roots. Copper has dose- depended effects on chlorophyll, proline, protein and abscisic acid level of sunflower (Helianthus annuus L.) seedlings. Thus, we assumed that copper levels increase above some critical points seedling growth get negative effects. This assumption is in line with previous findings.

  15. Proteomic Investigation of Protein Profile Changes and Amino Acid Residue Level Modification in Cooked Lamb Meat: The Effect of Boiling.

    PubMed

    Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

    2015-10-21

    Hydrothermal treatment (heating in water) is a common method of general food processing and preparation. For red-meat-based foods, boiling is common; however, how the molecular level effects of this treatment correlate to the overall food properties is not yet well-understood. The effects of differing boiling times on lamb meat and the resultant cooking water were here examined through proteomic evaluation. The longer boiling time was found to result in increased protein aggregation involving particularly proteins such as glyceraldehyde-3-phosphate dehydrogenase, as well as truncation in proteins such as in α-actinin-2. Heat-induced protein backbone cleavage was observed adjacent to aspartic acid and asparagine residues. Side-chain modifications of amino acid residues resulting from the heating, including oxidation of phenylalanine and formation of carboxyethyllysine, were characterized in the cooked samples. Actin and myoglobin bands from the cooked meat per se remained visible on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after significant cooking time. These proteins were also found to be the major source of observed heat-induced modifications. This study provides new insights into molecular-level modifications occurring in lamb meat proteins during boiling and a protein chemistry basis for better understanding the effect of this common treatment on the nutritional and functional properties of red-meat-based foods.

  16. Genome-Wide Association Study of CSF Levels of 59 Alzheimer's Disease Candidate Proteins: Significant Associations with Proteins Involved in Amyloid Processing and Inflammation

    PubMed Central

    Kauwe, John S. K.; Bailey, Matthew H.; Ridge, Perry G.; Perry, Rachel; Wadsworth, Mark E.; Hoyt, Kaitlyn L.; Staley, Lyndsay A.; Karch, Celeste M.; Harari, Oscar; Cruchaga, Carlos; Ainscough, Benjamin J.; Bales, Kelly; Pickering, Eve H.; Bertelsen, Sarah; Fagan, Anne M.; Holtzman, David M.; Morris, John C.; Goate, Alison M.

    2014-01-01

    Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer's disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC) and Alzheimer's Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10−10) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal

  17. Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

    PubMed

    Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

    2012-01-01

    Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

  18. Overexpression of hydroxynitrile lyase in cassava roots elevates protein and free amino acids while reducing residual cyanogen levels.

    PubMed

    Narayanan, Narayanan N; Ihemere, Uzoma; Ellery, Claire; Sayre, Richard T

    2011-01-01

    Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2-20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels.

  19. Elevated soluble HLA II protein levels in patients with alpha-1 antitrypsin deficiency with or without COPD.

    PubMed

    Li, Liping; Kueppers, Friedrich; Hildebrand, William; Buchli, Rico; Gaughan, John

    2012-08-01

    Elevated levels of human leukocyte antigen (HLA) proteins have been reported in several pathologic conditions that are associated with increased concentrations of white blood cells (e.g., infection, inflammation, and lymphoproliferative disorders). The mechanisms by which HLA proteins are solubilized from cell membranes are insufficiently understood. We hypothesized that HLA proteins may be cleaved from cell membranes by insufficiently inhibited leukocytic elastase, as expected in alpha-1 antitrypsin deficiency (A1ATD), resulting in elevated plasma levels of soluble HLA (sHLA) proteins. Using an enzyme-linked immunosorbent assay, we measured sHLA II levels in the peripheral blood of patients with A1ATD with or without co-existing chronic obstructive pulmonary disease (COPD), with COPD only, and in a control group. Mean (±SD) sHLA II plasma levels were 110 ± 200 pg/mL in patients with A1ATD and COPD (Group 1), 10 ± 30 pg/mL in patients with COPD without A1ATD (Group 2), 70 ± 90 pg/mL in patients with A1ATD without COPD (Group 3), and 10 ± 30 pg/mL in healthy donors (Group 4). Soluble HLA II plasma levels were significantly higher in Group 1 (P = .001) and Group 3 (P = .002) versus Group 4. Our preliminary results suggest that leukocytic elastase and probably other proteinases solubilize HLA proteins from cell membranes. This mechanism would operate in inflammation with elevated leukocytic elastase levels but more so with inflammation and A1ATD, where elastase would be insufficiently inhibited. If this mechanism is verified, plasma sHLA levels could potentially be used to measure cell damage due to proteinases and, therefore, for monitoring the therapeutic efficacy of alpha-1 antitrypsin (A1AT) augmentation therapy.

  20. Associations Between Common and Rare Exonic Genetic Variants and Serum Levels of 20 Cardiovascular-Related Proteins

    PubMed Central

    Solomon, Terry; Smith, Erin N.; Matsui, Hiroko; Braekkan, Sigrid K.; Wilsgaard, Tom; Njølstad, Inger; Mathiesen, Ellisiv B.; Hansen, John-Bjarne

    2016-01-01

    Background— Genetic variation can be used to study causal relationships between biomarkers and diseases. Here, we identify new common and rare genetic variants associated with cardiovascular-related protein levels (protein quantitative trait loci [pQTLs]). We functionally annotate these pQTLs, predict and experimentally confirm a novel molecular interaction, and determine which pQTLs are associated with diseases and physiological phenotypes. Methods and Results— As part of a larger case–control study of venous thromboembolism, serum levels of 51 proteins implicated in cardiovascular diseases were measured in 330 individuals from the Tromsø Study. Exonic genetic variation near each protein’s respective gene (cis) was identified using sequencing and arrays. Using single site and gene-based tests, we identified 27 genetic associations between pQTLs and the serum levels of 20 proteins: 14 associated with common variation in cis, of which 6 are novel (ie, not previously reported); 7 associations with rare variants in cis, of which 4 are novel; and 6 associations in trans. Of the 20 proteins, 15 were associated with single sites and 7 with rare variants. cis-pQTLs for kallikrein and F12 also show trans associations for proteins (uPAR, kininogen) known to be cleaved by kallikrein and with NTproBNP. We experimentally demonstrate that kallikrein can cleave proBNP (NTproBNP precursor) in vitro. Nine of the pQTLs have previously identified associations with 17 disease and physiological phenotypes. Conclusions— We have identified cis and trans genetic variation associated with the serum levels of 20 proteins and utilized these pQTLs to study molecular mechanisms underlying disease and physiological phenotypes. PMID:27329291

  1. The impact of a pure protein load on the glucose levels in type 1 diabetes patients treated with insulin pumps.

    PubMed

    Klupa, Tomasz; Benbenek-Klupa, Teresa; Matejko, Bartlomiej; Mrozinska, Sandra; Malecki, Maciej T

    2015-01-01

    We aimed to estimate the impact of ingestion of a pure protein load on the glucose levels in T1DM patients treated with insulin pumps. We examined 10 T1DM patients (6 females, mean age-32.3 years, mean HbA1c-6.8%) treated with insulin pumps equipped with a continuous glucose monitoring system (CGMS). In Phase I, baseline insulin infusion was optimized to minimize the differences in fasting glucose levels to less than 30 mg/dL between any two time points between 9 a.m. and 3 p.m. In Phase II, the patients were exposed to single pure protein load. CGMS record was performed and the glucose pattern was defined for 6 hours of each phase. The maximal glucose level increment was similar for the entire duration of the fasting and the protein load test (26.6 versus 27.6 mg/dL, resp., P < 0.78). There was only a borderline difference in change between baseline versus 6th hour glucose (12.5 and 19.0 mg/dL, P = 0.04). Glucose variability, assessed by standard deviation of mean glucose levels, was 36.4 and 37.9 mg/dL, respectively (P = 0.01). The administration of a pure protein load does not seem to have a clinically significant impact on glucose levels in T1DM patients treated with insulin pumps.

  2. The Impact of a Pure Protein Load on the Glucose Levels in Type 1 Diabetes Patients Treated with Insulin Pumps

    PubMed Central

    Klupa, Tomasz; Benbenek-Klupa, Teresa; Matejko, Bartlomiej; Mrozinska, Sandra; Malecki, Maciej T.

    2015-01-01

    We aimed to estimate the impact of ingestion of a pure protein load on the glucose levels in T1DM patients treated with insulin pumps. We examined 10 T1DM patients (6 females, mean age—32.3 years, mean HbA1c—6.8%) treated with insulin pumps equipped with a continuous glucose monitoring system (CGMS). In Phase I, baseline insulin infusion was optimized to minimize the differences in fasting glucose levels to less than 30 mg/dL between any two time points between 9 a.m. and 3 p.m. In Phase II, the patients were exposed to single pure protein load. CGMS record was performed and the glucose pattern was defined for 6 hours of each phase. The maximal glucose level increment was similar for the entire duration of the fasting and the protein load test (26.6 versus 27.6 mg/dL, resp., P < 0.78). There was only a borderline difference in change between baseline versus 6th hour glucose (12.5 and 19.0 mg/dL, P = 0.04). Glucose variability, assessed by standard deviation of mean glucose levels, was 36.4 and 37.9 mg/dL, respectively (P = 0.01). The administration of a pure protein load does not seem to have a clinically significant impact on glucose levels in T1DM patients treated with insulin pumps. PMID:25767510

  3. Variations in mRNA and protein levels of Ikaros family members in pediatric T cell acute lymphoblastic leukemia

    PubMed Central

    Mitchell, Julie L.

    2016-01-01

    Background Pediatric T cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous disease in which the cells share phenotypic characteristics with normal human thymocytes. The Ikaros family of transcription factors includes five members that are required for normal T cell development and are implicated in leukemogenesis. The goal of this work was to correlate the pattern of expression of Ikaros family members with the phenotype of the T-ALL cells. Methods We obtained twenty-four samples from pediatric T-ALL patients and used multi-parameter flow cytometry to characterize each sample, comparing the phenotype of the leukemic cells with normal human thymocytes. Then, we defined the expression levels of each Ikaros family member to determine whether the mRNA levels or splicing or protein levels were similar to the normal patterns seen during human T cell development. Results Multi-parameter analysis of the phenotype of T-ALL cells revealed that each patient’s cells were unique and could not be readily correlated with stages of T cell development. Similarly, the pattern of Ikaros expression varied among patients. In most patients, Ikaros mRNA was the dominant family member expressed, but some patients’ cells contained mostly Helios, Aiolos, or Eos mRNA. Despite that most patients had elevated mRNA levels of Ikaros family members and unique patterns of mRNA splicing, most patients had significantly reduced protein levels of Ikaros and Aiolos. Conclusions Our analysis of the cell phenotype and Ikaros expression levels in T-ALL cells revealed the extent of heterogeneity among patients. While it is rarely possible to trace leukemic cells to their developmental origin, we found distinct patterns of Ikaros family mRNA levels in groups of patients. Further, mRNA and protein levels of Ikaros and Aiolos did not correlate, indicating that mRNA and protein levels are regulated via distinct mechanisms. PMID:27826566

  4. Antidepressant effects of crocin and its effects on transcript and protein levels of CREB, BDNF, and VGF in rat hippocampus

    PubMed Central

    2014-01-01

    Background Antidepressants have been shown to affect levels of brain-derived neurotrophic factor (BDNF) and VGF (non-acronymic) whose transcriptions are dependent on cAMP response element binding protein (CREB) in long term treatment. The aim of this study was to verify the subacute antidepressant effects of crocin, an active constituent of saffron (Crocus sativus L.), and its effects on CREB, BDNF, and VGF proteins, transcript levels and amount of active, phosphorylated CREB (P-CREB) protein in rat hippocampus. Methods Crocin (12.5, 25, and 50 mg/kg), imipramine (10 mg/kg; positive control) and saline (1 mL/kg; neutral control) were administered intraperitoneally (IP) to male Wistar rats for 21 days. The antidepressant effects were studied using the forced swimming test (FST) on day 21 after injection. Protein expression and transcript levels of genes in the rat hippocampus were evaluated using western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), respectively. Results Crocin significantly reduced the immobility time in the FST. Western blot analysis showed that 25 and 50 mg/kg of crocin increased the levels of CREB and BDNF significantly and dose dependently. All doses of crocin increased the VGF levels in a dose-dependent manner. Levels of p-CREB increased significantly by 50 mg/kg dose of crocin. Only 12.5 mg/kg crocin could significantly increase the transcript levels of BDNF. No changes in CREB and VGF transcript levels were observed in all groups. Conclusions These results suggest that crocin has antidepressant-like action by increasing CREB, BDNF and VGF levels in hippocampus. PMID:24401376

  5. Design of a novel globular protein fold with atomic-level accuracy.

    PubMed

    Kuhlman, Brian; Dantas, Gautam; Ireton, Gregory C; Varani, Gabriele; Stoddard, Barry L; Baker, David

    2003-11-21

    A major challenge of computational protein design is the creation of novel proteins with arbitrarily chosen three-dimensional structures. Here, we used a general computational strategy that iterates between sequence design and structure prediction to design a 93-residue alpha/beta protein called Top7 with a novel sequence and topology. Top7 was found experimentally to be folded and extremely stable, and the x-ray crystal structure of Top7 is similar (root mean square deviation equals 1.2 angstroms) to the design model. The ability to design a new protein fold makes possible the exploration of the large regions of the protein universe not yet observed in nature.

  6. Growth and protein metabolism in red drum (Sciaenops ocellatus) larvae exposed to environmental levels of atrazine and malathion.

    PubMed

    McCarthy, Ian D; Fuiman, Lee A

    2008-07-30

    Contaminant exposure can affect development, growth, and behaviour of fish larvae, but its effect on rates of protein synthesis and protein degradation are not known. The aim of the present study was to examine the effects of a single pulsed dose aqueous exposure to environmentally realistic levels of two contaminants, atrazine (0, 40 and 80 microgl(-1)) and malathion (0, 1 and 10 microgl(-1)), on growth and protein synthesis in red drum (Sciaenops ocellatus) larvae. Growth was assessed in terms of increase in length, weight, and protein content over an 8-day period following exposure. Rates of protein synthesis were measured by the flooding dose technique 2, 4, and 8 days after initial exposure to each contaminant by bathing larvae in seawater containing L-[2,6-(3)H] phenylalanine. Exposure to atrazine had no effect on larval growth in length, but caused marginally significant declines in growth in weight (P=0.05) and protein content (P=0.06). However, protein synthesis rates were significantly higher for atrazine-exposed larvae on days 4 (P=0.04) and 8 (P=0.01), suggesting an increase in rates of protein degradation. On day 8, the efficiency with which synthesised proteins contributed to growth was significantly lower (P=0.04) in atrazine-exposed larvae. In contrast, malathion had no significant effects on growth in length or protein content, but there were significant decreases in growth in weight over 8 days. The only other significant effect of malathion was an increase in protein synthesis on day 2 for treated larvae relative to controls. Previous work [Alvarez, M.C., Fuiman, L.A., 2005. Environmental levels of atrazine and its degradation products impair survival skills and growth of red drum larvae. Aquat. Toxicol. 74, 229-241] reported hyperactivity and increased metabolic rate in larval red drum exposed to atrazine, indicating a clear energetic cost. Our results further emphasise the energetic cost of atrazine exposure through elevated rates of protein

  7. Dietary Japanese millet protein ameliorates plasma levels of adiponectin, glucose, and lipids in type 2 diabetic mice.

    PubMed

    Nishizawa, Naoyuki; Togawa, Tubasa; Park, Kyung-Ok; Sato, Daiki; Miyakoshi, Yo; Inagaki, Kazuya; Ohmori, Norimasa; Ito, Yoshiaki; Nagasawa, Takashi

    2009-02-01

    Millet is an important food crop in Asia and Africa, but the health benefits of dietary millet are little known. This study defined the effects of dietary Japanese millet on diabetic mice. Feeding of a high-fat diet containing Japanese millet protein concentrate (JMP, 20% protein) to type 2 diabetic mice for 3 weeks significantly increased plasma levels of adiponectin and high-density lipoprotein cholesterol (HDL cholesterol) and decreased the levels of glucose and triglyceride as compared to control. The starch fraction of Japanese millet had no effect on glucose or adiponectin levels, but the prolamin fraction beneficially modulated plasma glucose and insulin concentrations as well as adiponectin and tumor necrosis factor-alpha gene expression. Considering the physiological significance of adiponectin and HDL cholesterol levels in type 2 diabetes, insulin resistance, and cardiovascular disease, our findings imply that dietary JMP has the potential to ameliorate these diseases.

  8. Serum and tissue thiocyanate concentrations in growing pigs fed cassava peel or corn based diets containing graded protein levels.

    PubMed

    Tewe, O O

    1984-11-01

    Thiocyanate concentrations of serum, liver, kidney, spleen and longissimus dorsi were determined in 64 growing Large White x Landrace pigs offered 8 experimental isocaloric diets containing different levels of cassava peel and crude protein. Cassava peel increased serum thiocyanate on day 60 (P less than 0.01) and day 90 (P less than 0.01) of the trial, while the crude protein level increased it (P less than 0.05) on days 30 and 90, respectively. Interaction of the two factors was significant on day 30 (P less than 0.05) and day 90 (P less than 0.05). There was a correlation between cyanide intake and serum thiocyanate level. Coefficient of determination revealed that cyanide alone accounted for 28.5; 60.6 and 48.8% variation in serum thiocyanate on days 30, 60 and 90, respectively. Liver, spleen and longissimus dorsi thiocyanate were affected by dietary protein intake (P less than 0.05). Thiocyanate concentration was higher (P less than 0.05) on cassava peel diet. Generally, crude protein at 5% reduced organ and muscle thiocyanate concentrations. A diet containing 112.2-117.3 mg/kg hydrocyanic acid (HCN) affected serum but not organ and muscle thiocyanate in protein-sufficient diets.

  9. IFN-γ Directly Controls IL-33 Protein Level through a STAT1- and LMP2-dependent Mechanism*

    PubMed Central

    Kopach, Pavel; Lockatell, Virginia; Pickering, Edward M.; Haskell, Ronald E.; Anderson, Richard D.; Hasday, Jeffrey D.; Todd, Nevins W.; Luzina, Irina G.; Atamas, Sergei P.

    2014-01-01

    IL-33 contributes to disease processes in association with Th1 and Th2 phenotypes. IL-33 mRNA is rapidly regulated, but the fate of synthesized IL-33 protein is unknown. To understand the interplay among IL-33, IFN-γ, and IL-4 proteins, recombinant replication-deficient adenoviruses were produced and used for dual expression of IL-33 and IFN-γ or IL-33 and IL-4. The effects of such dual gene delivery were compared with the effects of similar expression of each of these cytokines alone. In lung fibroblast culture, co-expression of IL-33 and IFN-γ resulted in suppression of the levels of both proteins, whereas co-expression of IL-33 and IL-4 led to mutual elevation. In vivo, co-expression of IL-33 and IFN-γ in the lungs led to attenuation of IL-33 protein levels. Purified IFN-γ also attenuated IL-33 protein in fibroblast culture, suggesting that IFN-γ controls IL-33 protein degradation. Specific inhibition of caspase-1, -3, and -8 had minimal effect on IFN-γ-driven IL-33 protein down-regulation. Pharmacological inhibition, siRNA-mediated silencing, or gene deficiency of STAT1 potently up-regulated IL-33 protein expression levels and attenuated the down-regulating effect of IFN-γ on IL-33. Stimulation with IFN-γ strongly elevated the levels of the LMP2 proteasome subunit, known for its role in IFN-γ-regulated antigen processing. siRNA-mediated silencing of LMP2 expression abrogated the effect of IFN-γ on IL-33. Thus, IFN-γ, IL-4, and IL-33 are engaged in a complex interplay. The down-regulation of IL-33 protein levels by IFN-γ in pulmonary fibroblasts and in the lungs in vivo occurs through STAT1 and non-canonical use of the LMP2 proteasome subunit in a caspase-independent fashion. PMID:24619410

  10. Effects of Natural Osmolytes on the Protein Structure in Supercritical CO2: Molecular Level Evidence.

    PubMed

    Monhemi, Hassan; Housaindokht, Mohammad Reza; Nakhaei Pour, Ali

    2015-08-20

    Protein instability in supercritical CO2 limits the application of this green solvent in enzyme-catalyzed reactions. CO2 molecules act as a protein denaturant at high pressure under supercritical conditions. Here, for the first time, we show that natural osmolytes could stabilize protein conformation in supercritical CO2. Molecular dynamics simulation is used to monitor the effects of adding different natural osmolytes on the conformation and dynamics of chymotrypsin inhibitor 2 (CI2) in supercritical CO2. Simulations showed that CI2 is denatured at 200 bar in supercritical CO2, which is in agreement with experimental observations. Interestingly, the protein conformation remains native after addition of ∼1 M amino acid- and sugar-based osmolyte models. These molecules stabilize protein through the formation of supramolecular self-assemblies resulting from macromolecule-osmolyte hydrogen bonds. Nevertheless, trimethylamine N-oxide, which is known as a potent osmolyte for protein stabilization in aqueous solutions, amplifies protein denaturation in supercritical CO2. On the basis of our structural analysis, we introduce a new mechanism for the osmolyte effect in supercritical CO2, an "inclusion mechanism". To the best of our knowledge, this is the first study that introduces the application of natural osmolytes in a supercritical fluid and describes mechanistic insights into osmolyte action in nonaqueous media.

  11. Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations

    PubMed Central

    Hertig, Samuel

    2016-01-01

    Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein’s constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery—the fact that the two sites involved influence one another in a symmetrical manner—can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest. PMID:27285999

  12. The effect of varying protein levels on blood chemistry, food consumption, and behavior of captive seaducks

    USGS Publications Warehouse

    Wells-Berlin, A. M.; Perry, M.C.; Olsen, G.H.

    2005-01-01

    The Chesapeake Bay is a primary wintering area for scoters and the long-tailed ducks (Clangia hyemalis) that migrate along the Atlantic Flyway. Recently, the Chesapeake Bay had undergone an ecosystem shift and little is known about how this is affecting the seaduck populations. We are determining what are the preferred food sources of the seaducks wintering on the Bay and analyzing the factors influencing prey selection whether it is prey composition, energy assimilated, prey availability, or a combination of any or all of these factors. We have established a captive colony of surf (Melanitta perspicillata) and white-winged scoters (Melanitta fusca) as well as long-tailed ducks at Patuxent Wildlife Research Center to allow us to examine these factors in a more controlled environment. This project contains a multitude of experiments and the resultant data will be compiled into a compartmental model on the feeding ecology of seaducks wintering on the Bay. The first experiment entailed feeding groups of each species (four ducks per pen of equal sex ratio, if possible, and four pens per species) three diets varying in percent protein levels from November to February. Each diet was randomly assigned to each pen and the amount of food consumed was recorded each day. New feed was given when all existing food was consumed. Behavioral trials and blood profiles were completed on all study birds to determine the effects of the varying diets. There were no significant differences in food consumption, blood chemistry, and behavior detected at the 5% level among the diets for all three species of interest. There was a seasonal effect determined based on the food consumption data for white-winged scoters, but not for surf scoters or long-tailed ducks. The blood profiles of the surf scoters were compared to blood profiles of wild surf scoters and a there was no difference detected at the 5% level. As a health check of the ducks an aspergillosis test was run on the blood obtained

  13. Aging and exercise affect the level of protein acetylation and SIRT1 activity in cerebellum of male rats.

    PubMed

    Marton, Orsolya; Koltai, Erika; Nyakas, Csaba; Bakonyi, Tibor; Zenteno-Savin, Tania; Kumagai, Shuzo; Goto, Sataro; Radak, Zsolt

    2010-12-01

    Aging is associated with a gradual decline in cognitive and motor functions, the result of complex biochemical processes including pre- and posttranslational modifications of proteins. Sirtuins are NAD(+) dependent protein deacetylases. These enzymes modulate the aging process by lysine deacetylation, which alters the activity and stability of proteins. Exercise can increase mean life-span and improve quality of life. Data from our laboratories revealed that 4 weeks of treadmill running improves performance in the Morris Maze test for young (4 months, old) but not old (30 months, old) male rats, and the exercise could not prevent the age-associated loss in muscle strength assessed by a gripping test. The positive correlation between protein acetylation and the gripping test suggests that the age-dependent decrease in relative activity of SIRT1 in the cerebellum impairs motor function. Similarly to the acetylation level of total proteins, the acetylation of ά -tubulin is also increased with aging, while the effect of exercise training was not found to be significant. Moreover, the protein content of nicotinamide phosphoribosyltransferase, one of the key enzymes of NAD biosynthesis, decreased in the young exercise group. These data suggest that aging results in decreased specific activity of SIRT1 in cerebellum, which could lead to increased acetylation of protein residues, including ά-tubulin, that interfere with motor function.

  14. Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

    PubMed

    Sugiyama, Y; Sasajima, J; Mizukami, Y; Koizumi, K; Kawamoto, T; Ono, Y; Karasaki, H; Tanabe, H; Fujiya, M; Kohgo, Y

    2016-06-01

    The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

  15. Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation.

    PubMed

    Connolly, Erin L; Fett, Janette P; Guerinot, Mary Lou

    2002-06-01

    Iron, an essential nutrient, is not readily available to plants because of its low solubility. In addition, iron is toxic in excess, catalyzing the formation of hydroxyl radicals that can damage cellular constituents. Consequently, plants must carefully regulate iron uptake so that iron homeostasis is maintained. The Arabidopsis IRT1 gene is the major transporter responsible for high-affinity iron uptake from the soil. Here, we show that the steady state level of IRT1 mRNA was induced within 24 h after transfer of plants to iron-deficient conditions, with protein levels peaking 72 h after transfer. IRT1 mRNA and protein were undetectable 12 h after plants were shifted back to iron-sufficient conditions. Overexpression of IRT1 did not confer dominant gain-of-function enhancement of metal uptake. Analysis of 35S-IRT1 transgenic plants revealed that although IRT1 mRNA was expressed constitutively in these plants, IRT1 protein was present only in the roots when iron is limiting. Under these conditions, plants that overexpressed IRT1 accumulated higher levels of cadmium and zinc than wild-type plants, indicating that IRT1 is responsible for the uptake of these metals and that IRT1 protein levels are indeed increased in these plants. Our results suggest that the expression of IRT1 is controlled by two distinct mechanisms that provide an effective means of regulating metal transport in response to changing environmental conditions.

  16. Effect of dietary supplementation of omega-3 fatty acids and high levels of dietary protein on performance of sows.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was conducted to determine the effect of dietary supplementation of omega-3 fatty acids (O3FA), with or without high levels of dietary protein supplementation, on the performance of sows and their litters during first and subsequent parities. Sixty-four pregnant gilts with body weight (BW...

  17. Effect of dietary supplementation of omega-3 fatty acids and high levels of dietary protein on performance of sows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A study was conducted to determine the effect of dietary supplementation of omega-3 fatty acids (O3FA), with or without high levels of protein, on the performance of sows during first and subsequent parity. Sixty-four pregnant gilts with BW of 195.0 ± 2.1 kg and backfat (BF) thickness of 12.9 ± 0.2 ...

  18. Association of common C-protein (CRP) gene polymorphism with baseline plasma CRP levels and fenofibrate response: The GOLDN Study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    OBJECTIVE-C-reactive protein (CRP) is an inflammatory marker that contributes to the prediction of cardiovascular diseases (CVD). We investigated the influence of CRP polymorphisms on baseline CRP levels and fenofibrate-induced CRP changes in subjects with the metabolic syndrome (MetS). RESEARCH DES...

  19. TDP-43 aggregation mirrors TDP-43 knockdown, affecting the expression levels of a common set of proteins

    PubMed Central

    Prpar Mihevc, S.; Baralle, Marco; Buratti, Emanuele; Rogelj, Boris

    2016-01-01

    TDP-43 protein plays an important role in regulating transcriptional repression, RNA metabolism, and splicing. Typically it shuttles between the nucleus and the cytoplasm to perform its functions, while abnormal cytoplasmic aggregation of TDP-43 has been associated with neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). For the purpose of this study we selected a set of proteins that were misregulated following silencing of TDP-43 and analysed their expression in a TDP-43-aggregation model cell line HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L. Following TDP-43 sequestration in insoluble aggregates, we observed higher nuclear levels of EIF4A3, and POLDIP3β, whereas nuclear levels of DNMT3A, HNRNPA3, PABPC1 and POLDIP3α dropped, and cytoplasmic levels of RANBP1 dropped. In addition, immunofluorescence signal intensity quantifications showed increased nuclear expression of HNRNPL and YARS, and downregulation of cytoplasmic DPCD. Furthermore, cytoplasmic levels of predominantly nuclear protein ALYREF increased. In conclusion, by identifying a common set of proteins that are differentially expressed in a similar manner in these two different conditions, we show that TDP-43 aggregation has a comparable effect to TDP-43 knockdown. PMID:27665936

  20. Rhinacanthus nasutus Improves the Levels of Liver Carbohydrate, Protein, Glycogen, and Liver Markers in Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Visweswara Rao, Pasupuleti; Madhavi, K.; Dhananjaya Naidu, M.; Gan, Siew Hua

    2013-01-01

    The present study was designed to investigate the total carbohydrate, total protein, and glycogen levels in the liver and to measure functional liver markers such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in streptozotocin-(STZ-) induced diabetic rats after treatment with methanolic extract of Rhinacanthus nasutus (R. nasutus). The methanolic extract of R. nasutus was orally administered at 200 mg/kg/day while glibenclamide was administered at 50 mg/kg/day. All animals were treated for 30 days before being sacrificed. The amounts of carbohydrate, glycogen, proteins, and liver markers (AST and ALT) were measured in the liver tissue of the experimental animals. The levels of carbohydrate, glycogen, and proteins were significantly reduced in the diabetic rats but were augmented considerably after 30 days of R. nasutus treatment. The elevated AST and ALT levels in diabetic rats showed a significant decline after treatment with R. nasutus for 30 days. These results show that the administration of R. nasutus ameliorates the altered levels of carbohydrate, glycogen, proteins, and AST and ALT observed in diabetic rats and indicate that R. nasutus restores overall metabolism and liver function in experimental diabetic rats. In conclusion, the outcomes of the present study support the traditional belief that R. nasutus could ameliorate the diabetic state. PMID:24204387

  1. Nerve Growth Factor Increases mRNA Levels for the Prion Protein and the β -amyloid Protein Precursor in Developing Hamster Brain

    NASA Astrophysics Data System (ADS)

    Mobley, William C.; Neve, Rachael L.; Prusiner, Stanley B.; McKinley, Michael P.

    1988-12-01

    Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the β protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the β -protein precursor (β -PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and β -PP mRNAs. The increases in PrP and β -PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and β -PP mRNA levels. Increased PrP and β -PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and β -PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.

  2. A meta-analysis of potential risks of low levels of protein Z for diseases related to vascular thrombosis

    PubMed Central

    Sofi, Francesco; Cesari, Francesca; Abbate, Rosanna; Gensini, Gian Franco; Broze, George; Fedi, Sandra

    2013-01-01

    Summary The relationship between protein Z levels and thrombosis is controversial. We performed a systematic review and meta-analysis of the available studies to assess the association between protein Z and vascular thrombotic diseases. We conducted an electronic literature search through MedLine, Embase, Google Scholar, Web of Science, The Cochrane Library, bibliographies of retrieved articles and abstracts of congresses up to October, 2009. Studies were included if they analysed protein Z levels in patients with vascular thrombotic diseases. After the review process, 28 case-control studies (33 patient cohorts), including 4,218 patients with thrombotic diseases and 4,778 controls, were selected for analysis. The overall analysis using a random-effects model showed that low protein Z levels were associated with an increased risk of thrombosis (odds ratio [OR] 2.90, 95% confidence interval [CI] 2.05–4.12; p<0.00001). On subgroup analysis, a significant association was found between low protein Z levels and arterial vascular diseases (OR 2.67, 95%CI 1.60–4.48; p=0.0002), pregnancy complications (OR 4.17, 95%CI 2.31–7.52; p<0.00001), and venous thromboembolic diseases (OR 2.18, 95%CI 1.19–4.00; p=0.01). The results of this meta-analysis are consistent with a role for protein Z deficiency in thrombotic diseases, including arterial thrombosis, pregnancy complications and venous thromboembolism. PMID:20076855

  3. Enhanced levels of glutathione and protein glutathiolation in rat tongue epithelium during 4-NQO-induced carcinogenesis.

    PubMed

    Huang, Zhishan; Komninou, Despina; Kleinman, Wayne; Pinto, John T; Gilhooly, Elaine M; Calcagnotto, Ana; Richie, John P

    2007-04-01

    High glutathione (GSH) levels are commonly found in oral tumors and are thought to play an important role in tumorigenesis. While posttranslational binding of GSH to cellular proteins (protein glutathiolation) has recently been recognized as an important redox-sensitive regulatory mechanism, no data currently exist on this process during carcinogenesis. Our goal was to determine the effects of 4-nitroquinoline-N-oxide (4-NQO)-induced carcinogenesis on tongue levels of protein-bound and free GSH and related thiols in the rat. Male F-344 rats (6 weeks of age) were administered either 4-NQO (20 ppm) in drinking water or tap water alone (controls) for 8 weeks. Twenty-four weeks after cessation of 4-NQO, squamous cell carcinomas of the tongue were observed in all rats. The levels of both free and bound GSH in tumors, as well as in adjacent tissues, were 2- to 3-fold greater than in tongue epithelium from control rats (p < 0.05). Prior to tumor formation, at 8 weeks after cessation of 4-NQO, hyperplasia, dysplasia and carcinoma in situ were observed in 100%, 25% and 12.5% of 4-NQO-treated rats, respectively. At this early stage of carcinogenesis, levels of free and bound GSH were increased 50% compared with tongue tissues from control rats (p<0.05). Glutathione disulfide (GSSG) levels were also 2-fold greater in tongue tissues from 4-NQO treated vs. control rats (p<0.05). Altogether, these results suggest that protein glutathiolation, together with GSH and GSSG levels, are induced during oral carcinogenesis in the rat possibly as a result of enhanced levels of oxidative stress.

  4. Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

    SciTech Connect

    Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

  5. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

    PubMed Central

    2013-01-01

    Background The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. Results In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. Conclusion Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline. PMID:23802841

  6. Diverse Levels of Sequence Selectivity and Catalytic Efficiency of Protein-Tyrosine Phosphatases

    PubMed Central

    Selner, Nicholas G.; Luechapanichkul, Rinrada; Chen, Xianwen; Neel, Benjamin G.; Zhang, Zhong-Yin; Knapp, Stefan; Bell, Charles E.; Pei, Dehua

    2014-01-01

    The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences, but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >105-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >105-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3–18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A co-crystal structure of PTP1B bound with a nephrin pY1193 peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic

  7. Prebiotics affect nutrient digestibility but not faecal ammonia in dogs fed increased dietary protein levels.

    PubMed

    Hesta, M; Roosen, W; Janssens, G P J; Millet, S; De Wilde, R

    2003-12-01

    An increased protein content and less digestible protein sources in the diet can induce bad faecal odour. The present study investigated the effect of adding prebiotics to dog diets enriched with animal-derived protein sources on apparent digestibilities and faecal ammonia concentration. In three subsequent periods eight healthy beagle dogs were fed a commercial dog diet that was gradually supplemented by up to 50 % with meat and bone meal (MBM), greaves meal (GM) or poultry meal (PM) respectively. Afterwards, 3 % fructo-oligosaccharides or 3 % isomalto-oligosaccharides were substituted for 3 % of the total diet. Supplementation with animal-derived protein sources did not decrease the apparent N digestibility significantly but oligosaccharides did. On the other hand the bacterial N content (% DM) in the faeces was highest in the oligosaccharide groups followed by the protein-supplemented groups and lowest in the control groups. When the apparent N digestibility was corrected for bacterial N no significant differences were noted anymore except for the GM group where the corrected N digestibility was still lower after oligosaccharide supplementation. The amount of faecal ammonia was significantly increased by supplementing with protein or oligosaccharides in the MBM and GM groups but not in the PM group. When apparent N digestibility is interpreted, a correction for bacterial N should be taken into account, especially when prebiotics are added to the diet. Oligosaccharides did not reduce the faecal ammonia concentrations as expected.

  8. Atomic-level functional model of dengue virus Envelope protein infectivity

    PubMed Central

    Christian, Elizabeth A.; Kahle, Kristen M.; Mattia, Kimberly; Puffer, Bridget A.; Pfaff, Jennifer M.; Miller, Adam; Paes, Cheryl; Davidson, Edgar; Doranz, Benjamin J.

    2013-01-01

    A number of structures have been solved for the Envelope (E) protein from dengue virus and closely related flaviviruses, providing detailed pictures of the conformational states of the protein at different stages of infectivity. However, the key functional residues responsible for mediating the dynamic changes between these structures remain largely unknown. Using a comprehensive library of functional point mutations covering all 390 residues of the dengue virus E protein ectodomain, we identified residues that are critical for virus infectivity, but that do not affect E protein expression, folding, virion assembly, or budding. The locations and atomic interactions of these critical residues within different structures representing distinct fusogenic conformations help to explain how E protein (i) regulates fusion-loop exposure by shielding, tethering, and triggering its release; (ii) enables hinge movements between E domain interfaces during triggered structural transformations; and (iii) drives membrane fusion through late-stage zipper contacts with stem. These results provide structural targets for drug and vaccine development and integrate the findings from structural studies and isolated mutagenesis efforts into a cohesive model that explains how specific residues in this class II viral fusion protein enable virus infectivity. PMID:24158478

  9. Fully Blind Docking at the Atomic Level for Protein-Peptide Complex Structure Prediction.

    PubMed

    Yan, Chengfei; Xu, Xianjin; Zou, Xiaoqin

    2016-10-04

    Protein-peptide interactions play an important role in many cellular processes. In silico prediction of protein-peptide complex structure is highly desirable for mechanistic investigation of these processes and for therapeutic design. However, predicting all-atom structures of protein-peptide complexes without any knowledge about the peptide binding site and the bound peptide conformation remains a big challenge. Here, we present a docking-based method for predicting protein-peptide complex structures, referred to as MDockPeP, which starts with the peptide sequence and globally docks the all-atom, flexible peptide onto the protein structure. MDockPeP was tested on the peptiDB benchmarking database using both bound and unbound protein structures. The results show that MDockPeP successfully generated near-native peptide binding modes in 95.0% of the bound docking cases and in 92.2% of the unbound docking cases. The performance is significantly better than other existing docking methods. MDockPeP is computationally efficient and suitable for large-scale applications.

  10. Investigation into the Effects of Boron on Liver Tissue Protein Carbonyl, MDA, and Glutathione Levels in Endotoxemia.

    PubMed

    Balabanlı, Barbaros; Balaban, Tuba

    2015-10-01

    Endotoxin has been known to cause the formation and damage of free radical. The importance of boron for human life is increasing each passing day, and its consuming fields are continuing to expand due to the advances in science and technology. Therefore, in our study, we intended to investigate into the effects of boron on liver tissue oxidative events. Eighteen male Wistar albino rats were randomly separated into three equal groups in the experiments; control group, boron + endotoxin group, and endotoxin group. Dissolved in distilled water, boric acid (100 mg/kg) was administered to boron + endotoxin group via gavage procedure for 28 days. Only distilled water was administered to control and endotoxin groups via gavage procedure for 28 days. Then 4 mg/kg endotoxin (LPS; Escherichia coli 0111:B4) was intraperitoneally (ip) administered to boron + endotoxin and endotoxin groups on the 28th day. Sterile saline was injected into control group on the 28th day (ip). Malondialdehyde (MDA), which is the end product of lipid peroxidation in liver tissues, protein carbonyl compounds (PC), which are protein oxidization markers, and glutathione (GSH) levels were measured spectrophotometrically. The results were compared with Mann-Whitney U test. When boron + endotoxin group is compared with endotoxin group, PC levels of endotoxin group showed a significant increase. When GSH levels are compared, GSH level in boron + endotoxin group decreased according to endotoxin group. Variations among all groups in MDA levels were found to be statistically insignificant. We are of the opinion that endotoxin affects the proteins by forming free radicals, and boron may also cause the structural and/or functional changes in proteins in order to protect proteins from oxidization.

  11. A Phospholipid-Protein Complex from Antarctic Krill Reduced Plasma Homocysteine Levels and Increased Plasma Trimethylamine-N-Oxide (TMAO) and Carnitine Levels in Male Wistar Rats

    PubMed Central

    Bjørndal, Bodil; Ramsvik, Marie S.; Lindquist, Carine; Nordrehaug, Jan E.; Bruheim, Inge; Svardal, Asbjørn; Nygård, Ottar; Berge, Rolf K.

    2015-01-01

    Seafood is assumed to be beneficial for cardiovascular health, mainly based on plasma lipid lowering and anti-inflammatory effects of n-3 polyunsaturated fatty acids. However, other plasma risk factors linked to cardiovascular disease are less studied. This study aimed to penetrate the effect of a phospholipid-protein complex (PPC) from Antarctic krill on one-carbon metabolism and production of trimethylamine-N-oxide (TMAO) in rats. Male Wistar rats were fed isoenergetic control, 6%, or 11% PPC diets for four weeks. Rats fed PPC had reduced total homocysteine plasma level and increased levels of choline, dimethylglycine and cysteine, whereas the plasma level of methionine was unchanged compared to control. PPC feeding increased the plasma level of TMAO, carnitine, its precursors trimethyllysine and γ-butyrobetaine. There was a close correlation between plasma TMAO and carnitine, trimethyllysine, and γ-butyrobetaine, but not between TMAO and choline. The present data suggest that PPC has a homocysteine lowering effect and is associated with altered plasma concentrations of metabolites related to one-carbon metabolism and B-vitamin status in rats. Moreover, the present study reveals a non-obligatory role of gut microbiota in the increased plasma TMAO level as it can be explained by the PPC’s content of TMAO. The increased level of carnitine and carnitine precursors is interpreted to reflect increased carnitine biosynthesis. PMID:26371012

  12. A Phospholipid-Protein Complex from Antarctic Krill Reduced Plasma Homocysteine Levels and Increased Plasma Trimethylamine-N-Oxide (TMAO) and Carnitine Levels in Male Wistar Rats.

    PubMed

    Bjørndal, Bodil; Ramsvik, Marie S; Lindquist, Carine; Nordrehaug, Jan E; Bruheim, Inge; Svardal, Asbjørn; Nygård, Ottar; Berge, Rolf K

    2015-09-08

    Seafood is assumed to be beneficial for cardiovascular health, mainly based on plasma lipid lowering and anti-inflammatory effects of n-3 polyunsaturated fatty acids. However, other plasma risk factors linked to cardiovascular disease are less studied. This study aimed to penetrate the effect of a phospholipid-protein complex (PPC) from Antarctic krill on one-carbon metabolism and production of trimethylamine-N-oxide (TMAO) in rats. Male Wistar rats were fed isoenergetic control, 6%, or 11% PPC diets for four weeks. Rats fed PPC had reduced total homocysteine plasma level and increased levels of choline, dimethylglycine and cysteine, whereas the plasma level of methionine was unchanged compared to control. PPC feeding increased the plasma level of TMAO, carnitine, its precursors trimethyllysine and γ-butyrobetaine. There was a close correlation between plasma TMAO and carnitine, trimethyllysine, and γ-butyrobetaine, but not between TMAO and choline. The present data suggest that PPC has a homocysteine lowering effect and is associated with altered plasma concentrations of metabolites related to one-carbon metabolism and B-vitamin status in rats. Moreover, the present study reveals a non-obligatory role of gut microbiota in the increased plasma TMAO level as it can be explained by the PPC's content of TMAO. The increased level of carnitine and carnitine precursors is interpreted to reflect increased carnitine biosynthesis.

  13. Enhanced expression of glucose-regulated protein 78 correlates with malondialdehyde levels during the formation of liver cirrhosis in rats

    PubMed Central

    ZHANG, YUN; ZHANG, HUIYING; ZHAO, ZHONGFU; LV, MINLI; JIA, JIANTAO; ZHANG, LILI; TIAN, XIAOXIA; CHEN, YUNXIA; LI, BAOHONG; LIU, MINGSHE; HAN, DEWU; JI, CHENG

    2015-01-01

    The aim of the present study was to explore the role of glucose-regulated protein 78 (GRP78) in the development of liver cirrhosis promoted by intestinal endotoxemia in rats. Fifty-one male Wistar rats were randomly divided into the liver cirrhosis 4-week, 6-week and 8-week groups and the normal control group at each time point. Liver cirrhosis was induced by employing multiple pathogenic factors in the rats. Blood and liver tissues were collected. The levels of alanine aminotransferase (ALT), homocysteine, endotoxin and tumor necrosis factor-α (TNF-α) in the plasma, and TNF-α, malondialdehyde (MDA) and procollagen type III peptide (PIIIP) in the liver tissues were determined. The mRNA and protein expression levels of GRP78 in the liver were detected using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Morphological changes were observed through hematoxylin and eosin and van Gieson staining of the liver. Liver cirrhosis caused marked histopathological changes to the livers of the rats. Following significant increases in the levels of ALT, homocysteine, endotoxin and TNF-α in the plasma, and TNF-α, MDA and PIIIP in the liver tissues of all experimental groups with the progression of liver cirrhosis, the mRNA and protein expression levels of GRP78 also gradually increased. In addition, correlation analysis indicated that the enhanced expression of GRP78 correlated with the MDA levels of the rats during the formation of liver cirrhosis. PMID:26668603

  14. In utero exposure to benzene increases embryonic c-Myb and Pim-1 protein levels in CD-1 mice

    SciTech Connect

    Wan, Joanne; Winn, Louise M.

    2008-05-01

    Benzene is a known human leukemogen, but its role as an in utero leukemogen remains controversial. Epidemiological studies have correlated parental exposure to benzene with an increased incidence of childhood leukemias. We hypothesize that in utero exposure to benzene may cause leukemogenesis by affecting the embryonic c-Myb/Pim-1 signaling pathway and that this is mediated by oxidative stress. To investigate this hypothesis, pregnant CD-1 mice were treated with either 800 mg/kg of benzene or corn oil (i.p.) on days 10 and 11 of gestation and in some cases pretreated with 25 kU/kg of PEG-catalase. Phosphorylated and total embryonic c-Myb and Pim-1 protein levels were assessed using Western blotting and maternal and embryonic oxidative stress were assessed by measuring reduced to oxidized glutathione ratios. Our results show increased oxidative stress at 4 and 24 h after exposure, increased phosphorylated Pim-1 protein levels 4 h after benzene exposure, and increased Pim-1 levels at 24 and 48 h after benzene exposure. Embryonic c-Myb levels were elevated at 24 h after exposure. PEG-catalase pretreatment prevented benzene-mediated increases in embryonic c-Myb and Pim-1 protein levels, and benzene-induced oxidative stress. These results support a role for ROS in c-Myb and Pim-1 alterations after in utero benzene exposure.

  15. [Effect of Neowetadina and Polyvaccinum on serum levels of immunoglobulins and total proteins in calves during the industrial fattening process].

    PubMed

    Rzedzicki, J; Gliński, Z; Kaźmir, Z; Mikucki, J

    1986-01-01

    The usefulness of nonspecific stimulating preparations-Neowetadina (Biowet) and Poliwakcyna (Polfa) for stimulation of the serum level of immunoglobulins of the classes IgG, IgA and IgM and total protein was determined in calves of the black and white breed at the age of 9-10 weeks (average body weight 75-80 kg) over 8 weeks from the onset of industrial fattening in technological groups. The stimulants were given in single intramuscular injections at the following doses: 0.08 and 0.4 ml of Neowetadina per 1 kg of body weight, 0.4 ml of Poliwakcyna forte per 1 kg of body weight. The level of the particular classes of immunoglobulins in the calf sera was determined by radial immunodiffusion according to Mancini et al., modified by Fahey and McKelvey, and that of total protein by the biuret method. The serum of the IgG class immunoglobulins increased statistically significantly (P less than or equal to 0.05), starting from the 4th week after the administration of the preparations and reached its maximal value in the 5th week. However, the serum level of IgA increased in the 8th week of observation. Poliwakcyna and Neowetadina effected only slightly the increase of the serum level of IgM immunoglobulins. The preparations also stimulated the increase of total protein level in the blood serum of the calves.

  16. Altered Levels of Visinin-Like Protein 1 Correspond to Regional Neuronal Loss in Alzheimer Disease and Frontotemporal Lobar Degeneration.

    PubMed

    Kirkwood, Caitlin M; MacDonald, Matthew L; Schempf, Tadhg A; Vatsavayi, Anil V; Ikonomovic, Milos D; Koppel, Jeremy L; Ding, Ying; Sun, Mai; Kofler, Julia K; Lopez, Oscar L; Yates, Nathan A; Sweet, Robert A

    2016-02-01

    Recent studies have implicated the neuronal calcium-sensing protein visinin-like 1 protein (Vilip-1) as a peripheral biomarker in Alzheimer disease (AD), but little is known about expression of Vilip-1 in the brains of patients with AD. We used targeted and quantitative mass spectrometry to measure Vilip-1 peptide levels in the entorhinal cortex (ERC) and the superior frontal gyrus (SF) from cases with early to moderate stage AD, frontotemporal lobar degeneration (FTLD), and cognitively and neuropathologically normal elderly controls. We found that Vilip-1 levels were significantly lower in the ERC, but not in SF, of AD subjects compared to normal controls. In FTLD cases, Vilip-1 levels in the SF were significantly lower than in normal controls. These findings suggest a unique role for cerebrospinal fluid Vilip-1 as a biomarker of ERC neuron loss in AD.

  17. Impacts of acute imipramine treatment on plasma and brain amino acid metabolism in mice given graded levels of dietary chicken protein.

    PubMed

    Nagasawa, Mao; Murakami, Tatsuro; Tomonaga, Shozo; Sato, Mikako; Takahata, Yoshihisa; Morimatsu, Fumiki; Furuse, Mitsuhiro

    2012-12-01

    Several studies have shown a relationship between depression and animal protein intake. To evaluate whether the difference of dietary chicken protein levels induces an antidepressant-like effect and potentiates acute antidepressant effects, three levels of dietary chicken protein were used as the representative animal protein with imipramine used as the antidepressant. In addition, the effects of dietary chicken protein on brain metabolism were evaluated. Open field test (OFT) and forced swimming test (FST) were conducted on the 27th and 28th days, respectively. OFT and FST were not influenced by both imipramine and dietary protein levels. However, characteristic effects of imipramine treatment on brain monoamine metabolism were observed in the cerebral cortex and hypothalamus. In addition, dietary protein significantly increased taurine and L-ornithine levels even though these amino acids were not contained in the diets. In conclusion, the metabolism of several amino acids in the plasma and brain were altered by dietary chicken protein.

  18. Genes adopt non-optimal codon usage to generate cell cycle-dependent oscillations in protein levels

    PubMed Central

    Frenkel-Morgenstern, Milana; Danon, Tamar; Christian, Thomas; Igarashi, Takao; Cohen, Lydia; Hou, Ya-Ming; Jensen, Lars Juhl

    2012-01-01

    The cell cycle is a temporal program that regulates DNA synthesis and cell division. When we compared the codon usage of cell cycle-regulated genes with that of other genes, we discovered that there is a significant preference for non-optimal codons. Moreover, genes encoding proteins that cycle at the protein level exhibit non-optimal codon preferences. Remarkably, cell cycle-regulated genes expressed in different phases display different codon preferences. Here, we show empirically that transfer RNA (tRNA) expression is indeed highest in the G2 phase of the cell cycle, consistent with the non-optimal codon usage of genes expressed at this time, and lowest toward the end of G1, reflecting the optimal codon usage of G1 genes. Accordingly, protein levels of human glycyl-, threonyl-, and glutamyl-prolyl tRNA synthetases were found to oscillate, peaking in G2/M phase. In light of our findings, we propose that non-optimal (wobbly) matching codons influence protein synthesis during the cell cycle. We describe a new mathematical model that shows how codon usage can give rise to cell-cycle regulation. In summary, our data indicate that cells exploit wobbling to generate cell cycle-dependent dynamics of proteins. PMID:22373820

  19. Engineered mutations in fibrillin-1 leading to Marfan syndrome act at the protein, cellular and organismal levels.

    PubMed

    Zeyer, Karina A; Reinhardt, Dieter P

    2015-01-01

    Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated fibrillin-1.

  20. The pink-eyed dilution locus controls the biogenesis of melanosomes and levels of melanosomal proteins in the eye.

    PubMed

    Orlow, S J; Brilliant, M H

    1999-02-01

    The pink-eyed dilution (p) locus is known to control the quantity of melanin pigment made within melanocytes and retinal pigment epithelium (RPE) in the eye. We have examined the effects of several mutant allele combinations at the murine p locus on the number and morphology of melanosomes in choroidal melanocytes and RPE cells as well as on the levels of four proteins known to be present within melanosomes: tyrosinase, tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2) and lysosome-associated membrane protein-1 (LAMP-1). By electron microscopy, we observed a modest diminution in the size and number of choroidal melanosomes in pbs/pJ mice but a more dramatic decrease in the RPE in comparison with wild-type P/P mice. By contrast, a drastic reduction in melanosome size and number was present in the choroid and RPE of pun/pun and p6H/pcp mice, and in the RPE of p6H/pcp mice, melanosomes were essentially undetectable. In wild-type mice, levels of tyrosinase, TRP-1 and TRP-2 were high at birth and showed a second peak of expression at 10-14 days of age, declining to undetectable levels by 42 days. All three mutant allele combinations reduced the levels of these melanosomal proteins with the relative severity of effects being p6H/pcp>pun/pun>pbs/pJ. In the null p6H/pcp mice, levels of these proteins were extremely low at birth, no postnatal peak was observed, and levels declined to undetectable by 14 days. Levels of LAMP-1 in wild-type mice rose initially and then declined whereas in the mutant mice, levels decreased gradually from birth. Higher levels of LAMP-1 were observed in each of the mutants than in the wild-type mice at 21 days of age. Our results demonstrate that mutations at the p locus affect the size, number, shape and contents of melanosomes, implicating the p gene product in the normal biogenesis of this organelle.

  1. Three high-lysine mutations control the level of ATP-binding HSP70-like proteins in the maize endosperm.

    PubMed

    Marocco, A; Santucci, A; Cerioli, S; Motto, M; Di Fonzo, N; Thompson, R; Salamini, F

    1991-05-01

    The synthesis and deposition of seed storage proteins in maize are affected by several dominant and recessive mutants. The effect of three independent mutations, floury-2 (fl2), Defective endosperm-B30 (De-B30), and Mucronate (Mc), that reduce zein level in the endosperm were investigated. These mutations also control the level of b-70, a polypeptide bound to protein bodies, which is separable into the two isoforms b-70I and b-70II by two-dimensional gel electrophoresis. Both isoforms are overexpressed 10-fold in fl2; however, only b-70I is present in De-B30 and Mc, which contain an amount of total b-70 isoforms fivefold higher than in the wild type. Both b-70I and b-70II resemble heat shock protein (HSP70) in that they bind ATP, cross-react with anti-HSP antibodies, and have N-terminal sequence homology to HSP70. All maize protein body-located b-70 characteristics are typical of those of chaperone-like HSPs. A third protein, b-70III, similar in size to but slightly more acidic than b-70I and b-70II, also binds ATP and reacts with the same antibody, providing evidence for the presence in endosperm extracts of a cytosolic chaperone-like protein. The level of b-70III was not altered by the mutations studied. The results suggested that the repression effect of the three mutations on zein accumulation may be mediated by the alteration of a zein transport or zein assembly process involving b-70I and b-70II.

  2. Regional brain-derived neurotrophic factor mRNA and protein levels following transient forebrain ischemia in the rat.

    PubMed

    Kokaia, Z; Nawa, H; Uchino, H; Elmér, E; Kokaia, M; Carnahan, J; Smith, M L; Siesjö, B K; Lindvall, O

    1996-05-01

    Levels of BDNF mRNA and protein were measured in the rat brain using in situ hybridization and a two-site enzyme immunoassay. Under basal conditions, the highest BDNF concentration was found in the dentate gyrus (88 ng/g), while the levels in CA3 (50 ng/g), CA1 (18 ng/g) and parietal cortex (8 ng/g) were markedly lower. Following 10 min of forebrain ischemia, BDNF protein increased transiently in the dentate gyrus (to 124% of control at 6 h after the insult) and CA3 region (to 131% of control, at 1 week after the insult). In CA1 and parietal cortex, BDNF protein decreased to 73-75% of control at 24 h. In contrast, BDNF mRNA expression in dentate granule cells and CA3 pyramidal layer was transiently elevated to 287 and 293% of control, respectively, at 2 h, whereas no change was detected in CA1 or neocortex. The regional BDNF protein levels shown here correlate at least partly with regional differences in cellular resistance to ischemic damage, which is consistent with the hypothesis of a neuroprotective role of BDNF.

  3. Effects of dietary protein types on immune responses and levels of infection with Eimeria vermiformis in mice.

    PubMed

    Ford, J T; Wong, C W; Colditz, I G

    2001-02-01

    The present study reports the dietary effects of bovine alpha whey fraction, bovine casein and soy protein isolate on the immune responsiveness of C57BL/6J mice infected with Eimeria vermiformis. During the patent period, mice fed alpha whey fraction had significantly higher blood total white cell, CD4+ and CD8+ lymphocyte counts and higher Con A-stimulated IFN-gamma production by spleen cells than those fed other protein sources, but there was no significant difference in output of faecal oocysts. Casein-fed mice had significantly higher levels of Con A- stimulated IFN-gamma production and a lower output of faecal oocysts than soy-fed mice. The study demonstrated that dietary proteins have different impacts on immune responsiveness and level of parasitic infection in the gut; however, the mechanisms affecting level of infection are not clear. These effects appeared not to be solely related to nutritional properties of the diets. Further research into the underlying immune mechanisms and possible direct interactions between bioactive proteins and the parasite E. vermiformis should be fruitful.

  4. A constitutive damage specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells

    SciTech Connect

    Hirschfeld, S.; Levine, A.S.; Ozato, K.; Protic, M. )

    1990-05-01

    Using a DNA band shift assay, we have identified a DNA-binding protein complex in primate cells which is present constitutively and has a high affinity for UV-irradiated, double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin have higher levels of this damage-specific DNA-binding protein complex, suggesting that the signal for induction can either be damage to the DNA or interference with cellular DNA replication. Physiochemical modifications of the DNA and competition analysis with defined substrates suggest that the most probable target site for the damage-specific DNA-binding protein complex is a 6-4'-(pyrimidine-2'-one)-pyrimidine dimer: specific binding could not be detected with probes which contain -TT- cyclobutane dimers, and damage-specific DNA binding did not decrease after photoreactivation of UV-irradiated DNA. This damage-specific DNA-binding protein complex is the first such inducible protein complex identified in primate cells. Cells from patients with the sun-sensitive cancer-prone disease, xeroderma pigmentosum (group E), are lacking both the constitutive and the induced damage-specific DNA-binding activities. These findings suggest a possible role for this DNA-binding protein complex in lesion recognition and DNA repair of UV-light-induced photoproducts.

  5. The Prognostic Role of NEDD9 and P38 Protein Expression Levels in Urinary Bladder Transitional Cell Carcinoma

    PubMed Central

    Ali, Maged M.; El Shorbagy, Shereen; Abdelbary, Abeer M.; Abdelaziz, Lobna A.; Salim, Reham A.; Abdel Wahab, Khaled M.

    2017-01-01

    Background. The most common malignant tumor of the urinary bladder is transitional cell carcinoma (TCC). Neural precursor cell-expressed developmentally downregulated protein 9 (NEDD9) is found to be a cell adhesion mediator. P38 Mitogen-Activated Protein Kinase is a serine/threonine kinases member which can mediate carcinogenesis through intracellular signaling. Methods. To assess their prognostic role; NEDD9 and p38 protein were evaluated in sections from 50 paraffin blocks of TCC. Results. The high expressions of NEDD9 and p38 protein were significantly associated with grade, stage, distant metastasis (p < 0.001), number of tumors, lymph node metastasis, and tumor size (p < 0.001, 0.002; 0.018, <0.001; and 0.004, 0.007, respectively). High NEDD9 and p38 detection had a worse 3-year OS (p = 0.041 and <0.001, respectively). By multivariate analysis the NEDD9 and p38 protein expression levels and various clinicopathological criteria including gender, grade, stage of the tumor, and regional lymph node involvement were independent prognostic parameters of TCC of the urinary bladder patients' outcome. Conclusion. NEDD9 and p38 protein expressions were poor prognostic markers of TCC. PMID:28194179

  6. Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds.

    PubMed

    Wang, Wei; Scali, Monica; Vignani, Rita; Spadafora, Antonia; Sensi, Elisabetta; Mazzuca, Silvia; Cresti, Mauro

    2003-07-01

    The purpose of this research is to establish a routine procedure for the application of proteomic analysis to olive tree. Olive leaf tissue is notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds. We developed a protocol for isolating proteins suitable for two-dimensional electrophoresis (2-DE) from olive leaf. The remarkable characteristics of the protocol include: (i) additional grinding dry acetone powder of leaf tissue to a finer extent, (ii) after extensive organic solvent washes to remove pigments, lipids etc., using aqueous tricholoroacetic acid washes to remove water-soluble contaminants, and (iii) phenol extraction of proteins in the presence of sodium dodecyl sulfate. The final protein preparation is free of interfering compounds based on its well-resolved 2-DE patterns. The protocol can be completed within 3 h, and protein yield is approximately 2.49 mg.g(-1) of aged leaf. We also evaluated the protocol by immunoblotting with anti-tyrosinate alpha-tubulin antibody. To our knowledge, this is the first time that a protocol for protein extraction from olive leaf appears to give satisfactory and reproducible results. The protocol is expected to be applicable to other recalcitrant plant tissues and could be of interest to laboratories involved in plant proteomics.

  7. Heat Shock Partially Dissociates the Overlapping Modules of the Yeast Protein-Protein Interaction Network: A Systems Level Model of Adaptation

    PubMed Central

    Mihalik, Ágoston; Csermely, Peter

    2011-01-01

    Network analysis became a powerful tool giving new insights to the understanding of cellular behavior. Heat shock, the archetype of stress responses, is a well-characterized and simple model of cellular dynamics. S. cerevisiae is an appropriate model organism, since both its protein-protein interaction network (interactome) and stress response at the gene expression level have been well characterized. However, the analysis of the reorganization of the yeast interactome during stress has not been investigated yet. We calculated the changes of the interaction-weights of the yeast interactome from the changes of mRNA expression levels upon heat shock. The major finding of our study is that heat shock induced a significant decrease in both the overlaps and connections of yeast interactome modules. In agreement with this the weighted diameter of the yeast interactome had a 4.9-fold increase in heat shock. Several key proteins of the heat shock response became centers of heat shock-induced local communities, as well as bridges providing a residual connection of modules after heat shock. The observed changes resemble to a ‘stratus-cumulus’ type transition of the interactome structure, since the unstressed yeast interactome had a globally connected organization, similar to that of stratus clouds, whereas the heat shocked interactome had a multifocal organization, similar to that of cumulus clouds. Our results showed that heat shock induces a partial disintegration of the global organization of the yeast interactome. This change may be rather general occurring in many types of stresses. Moreover, other complex systems, such as single proteins, social networks and ecosystems may also decrease their inter-modular links, thus develop more compact modules, and display a partial disintegration of their global structure in the initial phase of crisis. Thus, our work may provide a model of a general, system-level adaptation mechanism to environmental changes. PMID:22022244

  8. Biotin deficiency and fatty liver and kidney syndrome in chicks given purified diets containing different fat and protein levels.

    PubMed

    Whitehead, C C; Bannister, D W; Evans, A J; Siller, W G; Wight, P A

    1976-01-01

    1. The occurence of biotin deficiency and fatty liver and kidney syndrome (FLKS) in chicks was studied using a 2x2x2x2 factorial-design experiment in which the variables were dietary biotin, fat and protein, and starvation. 2. The severity of biotin deficiency, using growth retardation and severity of dermal lesions as criteria, was least when the low-biotin diet also contained low levels of fat and protein. Addition of fat or protein increased the severity of the deficiency. Tissue fatty acid composition was affected by biotin deficiency only in those birds given the low-protein, low-fat diet. The main change was an increase in the ratio, 16:1 fatty acids :18:0 fatty acids. Plasma glucose and free fatty acid levels in non-fasted birds were unaffected by the dietary variables. 3. Mortality from FLKS with the diet containing low biotin, fat and protein levels was 52% at 28d, but was reduced or eliminated when the dietary level of any of these ingredients was increased. 4. Starvation considerably increased the incidnece of FLKS in the period immediately after fasting, and also affected plasma glucose and free fatty acid concentrations. Liver fatty acid composition, indicated an increase in the proportion of 18:0 at the expense of 16:1 and concentrations increased in proportion, at the expense of 18:0. 5. The relationship between biotin deficiency and FLKS, and a possible mechanism for the induction of FLKS by starvation are discussed.

  9. Lenalidomide affect expression level of cereblon protein in multiple myeloma cell line RPMI8226.

    PubMed

    Yang, D Y; Ren, J H; Guo, X N; Guo, X L; Cai, X Y; Guo, X F; Zhang, J N

    2015-10-29

    We investigated the mechanisms of action of immuno-modulatory drug (lenalidomide) on the protein expression of cereblon (CRBN) and their therapeutic targets in the multiple myeloma cell line RPMI8226. The multiple myeloma cell line RPMI8226 was cultured and treated with different concentrations of lenalidomide and bortezomib to determine the proliferation inhibition rate, apoptosis rate, and protein expression of CRBN. The results revealed that both lenalidomide and bortezomib inhibited the proliferation of RPMI8226 and promoted cell apoptosis. However, the protein expression of CRBN decreased signifi-cantly after treatment with lenalidomide, while bortezomib had no effect on the expression of CRBN. We confirmed that CRBN may be a target of lenalidomide.

  10. Levels of complement components, immunoglobulins and acute phase proteins in plasma during aging in Nigeria.

    PubMed

    Oyeyinka, G O; Salimonu, L S

    1999-01-01

    Plasma samples from Nigerians aged 6-95 years were examined for their content of complement components (C3, C4, factor B-Bf), immuloglobins (IgG, IgA, IgM IgD) and acute phase proteins (transferrin, albumin, C-reactive protein--CRP, alpha-2-macroglobulin). Albumin, was estimated colorimetrically and the other components by the single radial immunodiffusion techniques. No significant age-related changes in mean values of the four immunobulins and the four acute phase proteins could be demonstrated. Also, the mean values for C3 and Bf did not change significantly with age but C4 values rose significantly with increasing age (r -0.232: P < 0.01).

  11. Canavanine Alters ROS/RNS Level and Leads to Post-translational Modification of Proteins in Roots of Tomato Seedlings

    PubMed Central

    Krasuska, Urszula; Andrzejczak, Olga; Staszek, Paweł; Bogatek, Renata; Gniazdowska, Agnieszka

    2016-01-01

    Canavanine (CAN), a structural analog of arginine (Arg), is used as a selective inhibitor of inducible NOS in mammals. CAN is incorporated into proteins’ structure in the place of Arg, leading to the formation of aberrant compounds. This non-protein amino acid is found in legumes, e.g., Canavalia ensiformis (L.) DC. or Sutherlandia frutescens (L.) R.Br. and acts as a strong toxin against herbivores or plants. Tomato (Solanum lycopersicum L.) seedlings were treated for 24–72 h with CAN (10 or 50 μM) inhibiting root growth by 50 or 100%, without lethal effect. We determined ROS level/production in root extracts, fluorescence of DAF-FM and APF derivatives corresponding to RNS level in roots of tomato seedlings and linked CAN-induced restriction of root growth to the post-translational modifications (PTMs) of proteins: carbonylation and nitration. Both PTMs are stable markers of nitro-oxidative stress, regarded as the plant’s secondary response to phytotoxins. CAN enhanced H2O2 content and superoxide radicals generation in extracts of tomato roots and stimulated formation of protein carbonyl groups. An elevated level of carbonylated proteins was characteristic for the plants after 72 h of the culture, mainly for the roots exposed to 10 μM CAN. The proteolytic activity was stimulated by tested non-protein amino acid. CAN treatment led to decline of fluorescence of DAF-FM derivatives, and transiently stimulated fluorescence of APF derivatives. Short-term exposure of tomato seedlings to CAN lowered the protein nitration level. Activity of peroxidase, polyamine oxidase and NADPH oxidase, enzymes acting as modulators of H2O2 concentration and governing root architecture and growth were determined. Activities of all enzymes were stimulated by CAN, but no strict CAN concentration dependence was observed. We conclude, that although CAN treatment led to a decline in the nitric oxide level, PTMs observed in roots of plants exposed to CAN are linked rather to the

  12. Relationship of serum osteoprotegerin levels with coronary artery disease severity, left ventricular hypertrophy and C-reactive protein.

    PubMed

    Rhee, Eun-Jung; Lee, Won-Young; Kim, Se-Yeon; Kim, Byung-Jin; Sung, Ki-Chul; Kim, Bum-Su; Kang, Jin-Ho; Oh, Ki-Won; Oh, Eun-Sook; Baek, Ki-Hyun; Kang, Moo-Ii; Woo, Hee-Yeon; Park, Hyo-Soon; Kim, Sun-Woo; Lee, Man-Ho; Park, Jung-Roe

    2005-03-01

    OPG (osteoprotegerin) is an inhibitor of osteoclastogenesis and recent work suggests it has a role in atherosclerosis. Therefore we measured serum OPG levels in patients with coronary artery disease, compared the serum OPG levels among the different groups according to the number of stenotic vessels and determined whether there was any correlation with aortic calcification, LV (left ventricular) mass index and serum CRP (C-reactive protein) levels. Subjects (n=100; mean age, 57 years) who underwent coronary angiograms were enrolled. Blood pressure, body mass index, fasting blood glucose, lipid profiles and CRP levels were measured and the LV mass indices were calculated using ECGs. Serum OPG levels were measured by ELISA. The presence of calcification in the aortic notch was checked by a chest X-ray. The subjects were divided into four groups according to the number of stenotic vessels. The mean serum OPG levels increased significantly as the number of stenotic vessels increased, and the mean serum OPG levels were higher in the group with three-vessel disease compared with the groups with no- or one-vessel disease. The mean serum CRP level was significantly higher in the group with three-vessel disease compared with the groups with no-, one- and two-vessel disease. Age and LV mass index showed significant positive correlations with serum OPG levels, although significance was lost after an adjustment for age. Serum CRP levels were positively correlated with serum OPG levels even after an adjustment for age. There were no differences in serum OPG levels according to the presence of fasting hyperglycaemia or aortic calcification. In conclusion, serum OPG level was related to the severity of stenotic coronary arteries and serum CRP levels. LV mass indices showed no significant correlation with OPG levels. The precise mechanism for the role of OPG in atherosclerosis needs to be investigated further.

  13. Poly r(C) binding protein (PCBP) 1 expression is regulated at the post-translation level in thyroid carcinoma

    PubMed Central

    Zhang, Ming-Peng; Zhang, Wei-San; Tan, Jin; Zhao, Ming-Hui; Lian, Lin-Juan; Cai, Jie

    2017-01-01

    Poly r(C) binding protein (PCBP) 1 or heterogeneous ribonucleoprotein (hnRNP) E1 is a RNA binding protein that plays a vital role in a wide variety of biological processes. PCBP1 has been shown to function as a tumor suppressor by negatively regulating translation of pro-metastatic proteins in different cancers. Loss of PCBP1 expression or its Akt2-mediated phosphorylation at serine 43 residue has both been indicated to de-repress its regulation of EMT inducer proteins. Our previous work has established that PCBP1 functions as a tumor suppressor in thyroid cancer, where its translation is inhibited by microRNA-490-3p. Here we show that thyroid cancer patients can be divided into 2 cohorts based on miR-490-3p expression and PCBP1 mRNA expression-one cohort with high PCBP1 mRNA expression and basal miR-490-3p expression and a second cohort with low PCBP1 mRNA expression and high miR-490-3p expression. However, PCBP1 protein expression is also downregulated in the cohort with high PCBP1 mRNA expression, with expression levels similar to what is observed in patients with the low PCBP1 mRNA expression. Our analysis shows that PCBP1 mRNA is actively translated in patients with high PCBP1 mRNA expression, but that the protein is post translationally degraded by the proteasome machinery. Our results thus elucidate a novel mechanism responsible for down regulation of PCBP1 expression in thyroid cancer. It will be important in future to identify the mechanism that causes degradation of PCBP1 protein and to identify if similar mechanisms are active in other tumors characterized by low PCBP1 protein expression.

  14. Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level

    NASA Astrophysics Data System (ADS)

    Ray, Sujay

    Guanine-rich nucleic acid (DNA/RNA) sequences can form non-canonical secondary structures, known as G-quadruplex (GQ). Numerous in vivo and in vitro studies have demonstrated formation of these structures in telomeric and non-telomeric regions of the genome. Telomeric GQs protect the chromosome ends whereas non-telomeric GQs either act as road blocks or recognition sites for DNA metabolic machinery. These observations suggest the significance of these structures in regulation of different metabolic processes, such as replication and repair. GQs are typically thermodynamically more stable than the corresponding Watson-Crick base pairing formed by G-rich and C-rich strands, making protein activity a crucial factor for their destabilization. Inside the cell, GQs interact with different proteins and their enzymatic activity is the determining factor for their stability. We studied interactions of several proteins with GQs to understand the underlying principles of protein-GQ interactions using single-molecule FRET and other biophysical techniques. Replication Protein-A (RPA), a single stranded DNA (ssDNA) binding protein, is known to posses GQ unfolding activity. First, we compared the thermal stability of three potentially GQ-forming DNA sequences (PQS) to their stability against RPA-mediated unfolding. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQS in the genome. The thermal stability of these structures do not necessarily correlate with their stability against protein-mediated unfolding. We conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. To determine the critical structural factors that influence protein-GQ interactions we studied two groups of GQ structures that have systematically varying loop lengths and number of G-tetrad layers. We

  15. High levels of corticotropin-releasing factor (CRF) are inversely correlated with low levels of maternal CRF-binding protein in pregnant women with pregnancy-induced hypertension.

    PubMed

    Petraglia, F; Florio, P; Benedetto, C; Gallo, C; Woods, R J; Genazzani, A R; Lowry, P J

    1996-02-01

    Corticotropin-releasing factor-binding protein (CRF-BP) is suggested to play a role in modulating the activity of the hypothalamus-pituitary-adrenal axis during pregnancy, counteracting the actions of circulating or locally acting CRF. The aim of the present study was to evaluate whether maternal levels of CRF-BP and CRF are modified in pregnant women with a high risk of developing pregnancy-induced hypertension (n = 21). A group of nine patients developed the disease between 25-35 weeks gestation, and sequential blood samples were taken every 5 weeks throughout the pregnancy. As a control group, healthy pregnant women were studied (n = 9) using the same protocol; a group of women with pregnancy-induced hypertension (n = 5) was studied starting from the time of diagnosis. In a subgroup of patients (n = 10), CRF-BP and CRF levels were studied after 5 weeks of antihypertensive treatment. Levels of CRF-BP were determined using a specific RIA, whereas CRF was evaluated by a two-site immunoradiometric assay. In patients at risk, circulating levels of CRF-BP followed the same pattern as that in healthy controls, showing a significant decrease at term (36-40 weeks; P < 0.05). A significant and progressive increase in plasma CRF levels was observed in both groups of pregnant women; the highest values were found at term (P < 0.01). In the nine patients who developed pregnancy-induced hypertension, maternal levels of CRF-BP at the onset of signs and symptoms were lower than control values, and CRF levels were significantly higher at the onset of the disease (P < 0.01). Similarly, in these hypertensive patients studied at the time of hospitalization, CRF-BP levels were lower whereas those of CRF were higher than levels in healthy patients (P < 0.01). No effect of antihypertensive therapy on either CRF-BP or CRF levels was observed. The present study shows an inverse correlation between reduced plasma CRF-BP levels and increased CRF levels in the maternal circulation of

  16. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer.

    PubMed

    Chen, Yi; Pirisi, Lucia; Creek, Kim E

    2013-09-01

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer.

  17. The induction levels of heat shock protein 70 differentiate the vulnerabilities to mutant huntingtin among neuronal subtypes.

    PubMed

    Tagawa, Kazuhiko; Marubuchi, Shigeki; Qi, Mei-Ling; Enokido, Yasushi; Tamura, Takuya; Inagaki, Reina; Murata, Miho; Kanazawa, Ichiro; Wanker, Erich E; Okazawa, Hitoshi

    2007-01-24

    The reason why vulnerabilities to mutant polyglutamine (polyQ) proteins are different among neuronal subtypes is mostly unknown. In this study, we compared the gene expression profiles of three types of primary neurons expressing huntingtin (htt) or ataxin-1. We found that heat shock protein 70 (hsp70), a well known chaperone molecule protecting neurons in the polyQ pathology, was dramatically upregulated only by mutant htt and selectively in the granule cells of the cerebellum. Granule cells, which are insensitive to degeneration in the human Huntington's disease (HD) pathology, lost their resistance by suppressing hsp70 with siRNA, whereas cortical neurons, affected in human HD, gained resistance by overexpressing hsp70. This indicates that induction levels of hsp70 are a critical factor for determining vulnerabilities to mutant htt among neuronal subtypes. CAT (chloramphenicol acetyltransferase) assays showed that CBF (CCAAT box binding factor, CCAAT/enhancer binding protein zeta) activated, but p53 repressed transcription of the hsp70 gene in granule cells. Basal and mutant htt-induced expression levels of p53 were remarkably lower in granule cells than in cortical neurons, suggesting that different magnitudes of p53 are linked to distinct induction levels of hsp70. Surprisingly, however, heat shock factor 1 was not activated in granule cells by mutant htt. Collectively, different levels of hsp70 among neuronal subtypes might be involved in selective neuronal death in the HD pathology.

  18. Levels of the growth-associated protein GAP-43 are selectively increased in association cortices in schizophrenia

    PubMed Central

    Perrone-Bizzozero, Nora I.; Sower, Angela C.; Bird, Edward D.; Benowitz, Larry I.; Ivins, Kathryn J.; Neve, Rachael L.

    1996-01-01

    The pathophysiology of schizophrenia may involve perturbations of synaptic organization during development. The presence of cytoarchitectural abnormalities that may reflect such perturbations in the brains of patients with this disorder has been well-documented. Yet the mechanistic basis for these features of the disorder is still unknown. We hypothesized that altered regulation of the neuronal growth-associated protein GAP-43, a membrane phosphoprotein found at high levels in the developing brain, may play a role in the alterations in brain structure and function observed in schizophrenia. In the mature human brain, GAP-43 remains enriched primarily in association cortices and in the hippocampus, and it has been suggested that this protein marks circuits involved in the acquisition, processing, and/or storage of new information. Because these processes are known to be altered in schizophrenia, we proposed that GAP-43 levels might be altered in this disorder. Quantitative immunoblots revealed that the expression of GAP-43 is increased preferentially in the visual association and frontal cortices of schizophrenic patients, and that these changes are not present in other neuropsychiatric conditions requiring similar treatments. Examination of the levels of additional markers in the brain revealed that the levels of the synaptic vesicle protein synaptophysin are reduced in the same areas, but that the abundance of the astrocytic marker of neurodegeneration, the glial fibrillary acidic protein, is unchanged. In situ hybridization histochemistry was used to show that the laminar pattern of GAP-43 expression appears unaltered in schizophrenia. We propose that schizophrenia is associated with a perturbed organization of synaptic connections in distinct cortical associative areas of the human brain, and that increased levels of GAP-43 are one manifestation of this dysfunctional organization. PMID:8943081

  19. Electroacupuncture Improves Insulin Resistance by Reducing Neuroprotein Y/Agouti-Related Protein Levels and Inhibiting Expression of Protein Tyrosine Phosphatase 1B in Diet-induced Obese Rats.

    PubMed

    Liu, Xia; He, Jun-Feng; Qu, Ya-Ting; Liu, Zhi-Jun; Pu, Qing-Yang; Guo, Sheng-Tong; Du, Jia; Jiang, Peng-Fei

    2016-04-01

    Electroacupuncture (EA) has been shown to exert beneficial effects on obesity, but the mechanism is unclear. This study investigated the effects of EA on diet-induced obese (DIO) rats. Fifty male Sprague-Dawley rats were randomly divided into low-fat diet (LFD, 10 rats) and high-fat diet (HFD, 40 rats) groups. After the DIO models had been established, successful model rats were randomly divided into HFD, EA, and orlistat (OLST) groups. The EA group received EA at Zusanli (ST36) and Quchi (LI11) for 20 minutes once per day for 28 days. The OLST group was treated with orlistat by gavage. The body weight, homeostasis model assessment-insulin resistance index, adipocyte diameters, and neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B levels were significantly lower in the EA group than in the HFD group. The rats of the OLST group showed watery stools and yellow hairs whereas those of the EA group had regular stools and sleek coats. The effect of EA on weight loss may be related to improved insulin resistance caused by changes in the adipocyte size and by reductions in the expressions of neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B. This study indicates that EA may be a better method of alternative therapy for treating obesity and other metabolic diseases.

  20. Xylella fastidiosa infection of grapevines affects xylem levels of phenolic compounds and pathogenesis-related proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pierce’s disease (PD), caused by the xylem-dwelling pathogen Xylella fastidiosa (X.f.), is a serious threat to grape production. The effects of X.f. infection six months post-inoculation on defense-associated proteins and phenolic compounds found in xylem sap and tissue were evaluated. Defense-assoc...

  1. Protein expression levels in the medullary visceral zone of rats with subarachnoid hemorrhage.

    PubMed

    Sun, L H; Xing, L F; Zhang, G H; Pan, S Y

    2015-08-03

    We investigated protein expression in the medullary visceral zone (MVZ) of rats with multiple-organ dysfunction syndrome (MODS) caused by subarachnoid hemorrhage (SAH) to discuss the possible regulatory mechanism of the MVZ in the course of SAH-induced MODS. A SAH-induced MODS model was established in rats by injecting arterial blood into the Willis' circle. Protein expression in the MVZ was analyzed by immunohistochemistry assay. Protein expression in the MVZ peaked 24-36 h after SAH, and was significantly higher than in the control and sham operation groups. Organs at each time point exhibited inflammatory injuries to varying degrees after SAH, which reached a maximum at 24-36 h. Incidences of systemic inflammatory response syndrome and MODS were 100 and 71.67%, respectively, after SAH. There is a consistency between MVZ protein expression and inflammatory changes in each organ after SAH. This prompts the suggestion that the MVZ may be one of the direct regulative centers in SAH-induced MODS, and may be involved in the functional regulation of the surrounding organs after SAH.

  2. Methylation potential associated with diet, genotype, protein, and metabolite levels in the Delta Obesity Vitamin Study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Micronutrient research typically focuses on analyzing the effects of single or a few nutrients on health by analyzing a limited number of biomarkers. The observational study described here analyzed micronutrients, plasma proteins, dietary intakes, and genotype using a systems approach. Participants ...

  3. Effects of temperature and dietary protein level on hepatic oxidative status of Senegalese sole juveniles (Solea senegalensis).

    PubMed

    Castro, C; Pérez-Jiménez, A; Guerreiro, I; Peres, H; Castro-Cunha, M; Oliva-Teles, A

    2012-11-01

    Effects of 55 and 45% dietary protein levels (55P and 45P diets, respectively) and temperature (12 and 18 °C) on hepatic activity of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase (GR), glucose-6-phosphate dehydrogenase and lipid peroxidation (LPO) levels of Solea senegalensis juveniles were studied. Further, effects of acute thermal shocks provoked by a drop (18 °C to 12 °C) or a rise (12 °C to 18 °C) of water temperature on sole oxidative state was also evaluated. Dietary protein reduction increased LPO levels though no major alterations were found on antioxidant enzyme activities between dietary treatments. At 12 °C GR activity was higher and SOD activity was lower than 18 °C but LPO levels were not affected. In both thermal shock cases, LPO levels increased in 55P group, probably due to insufficient antioxidant enzyme activation. In contrast, fish of 45P group under acute exposition to warmer and colder temperature exhibited no substantial changes and a significant decrease on LPO levels, respectively, along with no major changes in antioxidant enzymes. Overall, results suggest that independently of rearing temperatures 45P group was more susceptible to oxidative stress than 55P group. Thermal shock either due to rise or drop of temperature seemed to induce oxidative stress in 55P group.

  4. Lipid and protein oxidation of chicken breast rolls as affected by dietary oxidation levels and packaging.

    PubMed

    Xiao, Shan; Zhang, Wan Gang; Lee, Eun Joo; Ma, Chang Wei; Ahn, Dong U

    2011-05-01

    The objective of this study was to determine the effects of dietary treatment and packaging on the oxidative stability of breast rolls. A total of 120 4-wk-old broiler chickens were randomly assigned to control, oxidized diet (5% oxidized oil, PV = 100), or antioxidants-added diet (500 IU vitamin E + 200 ppm BHA) and fed for 2 wk. Breast muscles were separated from the carcasses and breast rolls were prepared. The rolls were cooked in a smoke house (85 °C) to an internal temperature of 74 °C, cooled, sliced to 2-cm thick pieces, individually packaged in oxygen permeable bags or vacuum-packaged in oxygen impermeable bags, and stored in a 4 °C cold room for 7 d. Lipid, protein oxidation and volatiles were determined at 1, 4, and 7 d of storage. Dietary supplementation of antioxidants significantly reduced lipid oxidation (TBARS) and protein oxidation (carbonyls) in breast rolls, and the effect of dietary antioxidants on lipid oxidation was more pronounced than protein oxidation. Chicken breast rolls from antioxidants treatment group produced significantly lower amounts of hexanal and pentanal than those from control and oxidized oil treatments (P < 0.05). However, dietary oxidized oil did not increase lipid and protein oxidation in breast rolls. Vacuum-packaging significantly delayed the onset of lipid oxidation and protein oxidation in chicken rolls during 7-day refrigerated storage (P < 0.05). Therefore, it is suggested that appropriate use of dietary supplementation of antioxidants in combination with packaging could minimize lipid oxidation in chicken breast rolls.

  5. Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae

    PubMed Central

    Tavakoli, Minoo; Bouzari, Saeed; Siadat, Seyed Davar; Najar Peerayeh, Shahin; Jafari, Anis

    2015-01-01

    Background: Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis. Objectives: In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study. Materials and Methods: At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting. Results: The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration. Conclusions: The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway. PMID:26034537

  6. Identification of endogenously S-nitrosylated proteins in Arabidopsis plantlets: effect of cold stress on cysteine nitrosylation level.

    PubMed

    Puyaubert, Juliette; Fares, Abasse; Rézé, Nathalie; Peltier, Jean-Benoît; Baudouin, Emmanuel

    2014-02-01

    S-nitrosylation is a nitric oxide (NO)-based post-translational modification regulating protein function and signalling. We used a combination between the biotin switch method and labelling with isotope-coded affinity tag to identify endogenously S-nitrosylated peptides in Arabidopsis thaliana proteins extracted from plantlets. The relative level of S-nitrosylation in the identified peptides was compared between unstressed and cold-stress seedlings. We thereby detected 62 endogenously nitrosylated peptides out of which 20 are over-nitrosylated following cold exposure. Taken together these data provide a new repertoire of endogenously S-nitrosylated proteins in Arabidopsis with cysteine S-nitrosylation site. Furthermore they highlight the quantitative modification of the S-nitrosylation status of specific cysteine following cold stress.

  7. Rice protein improves adiposity, body weight and reduces lipids level in rats through modification of triglyceride metabolism

    PubMed Central

    2012-01-01

    Background To elucidate whether rice protein can possess a vital function in improving lipids level and adiposity, the effects of rice proteins extracted by alkaline (RP-A) and α-amylase (RP-E) on triglyceride metabolism were investigated in 7-week-old male Wistar rats fed cholesterol-enriched diets for 2 weeks, as compared with casein (CAS). Results Compared with CAS, plasma concentrations of glucose and lipids were significantly reduced by RP-feeding (P < 0.05), as well as hepatic accumulation of lipids (P < 0.05). RP-A and RP-E significantly depressed the hepatic activities of fatty acid synthase (FAS), glucose 6-phosphate dehydrogenase (G6PD) and malate dehydrogenase (MDH) (P < 0.05), whereas the activities of lipoprotein lipase (PL) and hepatic lipase (HL) were significantly stimulated (P < 0.05), as compared to CAS. Neither lipids level nor activities of enzymes were different between RP-A and RP-E (P > 0.05). There was a significant positive correlation between protein digestibility and deposit fat (r = 0.8567, P < 0.05), as well as the plasma TG concentration (r = 0.8627, P < 0.05). Conclusions The present study demonstrates that rice protein can modify triglyceride metabolism, leading to an improvement of body weight and adiposity. Results suggest that the triglyceride-lowering action as well as the potential of anti-adiposity induced by rice protein is attributed to upregulation of lipolysis and downregulation of lipogenesis, and the lower digestibility of rice protein may be the main modulator responsible for the lipid-lowering action. PMID:22330327

  8. From transcriptomic to protein level changes in TDP-43 and FUS loss-of-function cell models.

    PubMed

    Colombrita, Claudia; Onesto, Elisa; Buratti, Emanuele; de la Grange, Pierre; Gumina, Valentina; Baralle, Francisco E; Silani, Vincenzo; Ratti, Antonia

    2015-12-01

    The full definition of the physiological RNA targets regulated by TDP-43 and FUS RNA-binding proteins (RBPs) represents an important issue in understanding the pathogenic mechanisms associated to these two proteins in amyotrophic lateral sclerosis and frontotemporal dementia. In the last few years several high-throughput screenings have generated a plethora of data, which are difficult to compare due to the different experimental designs and models explored. In this study by using the Affymetrix Exon Arrays, we were able to assess and compare the effects of both TDP-43 and FUS loss-of-function on the whole transcriptome using the same human neuronal SK-N-BE cell model. We showed that TDP-43 and FUS depletion induces splicing and gene expression changes mainly distinct for the two RBPs, although they may regulate common pathways, including neuron differentiation and cytoskeleton organization as evidenced by functional annotation analysis. In particular, TDP-43 and FUS were found to regulate splicing and expression of genes related to neuronal (SEPT6, SULT4A1, TNIK) and RNA metabolism (DICER, ELAVL3/HuC, POLDIP3). Our extended analysis at protein level revealed that these changes have also impact on the protein isoform ratio and content, not always in a direct correlation with transcriptomic data. Contrarily to a loss-of-function mechanism, we showed that mutant TDP-43 proteins maintained their splicing activity in human ALS fibroblasts and experimental cell lines. Our findings further contribute to define the biological functions of these two RBPs in physiological and disease state, strongly encouraging the evaluation of the identified transcriptomic changes at protein level in neuronal experimental models.

  9. Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

    PubMed Central

    de Moraes, Vanessa C S; Bernardinelli, Emanuele; Zocal, Nathalia; Fernandez, Jhonathan A; Nofziger, Charity; Castilho, Arthur M; Sartorato, Edi L; Paulmichl, Markus; Dossena, Silvia

    2016-01-01

    Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants. PMID:26752218

  10. Low Serum Levels of Prealbumin, Retinol Binding Protein, and Retinol Are Frequent in Adult Type 1 Diabetic Patients

    PubMed Central

    Bolado, Federico; Goñi, María José; Tamayo, Ibai; Ibáñez, Berta; Prieto, Carlos

    2016-01-01

    Aim. To determine the serum prealbumin (PA), retinol binding protein (RBP), and retinol levels in adult patients with type 1 diabetes (T1D) and to analyze some factors related to those levels. Methods. A total of 93 patients (47 women) were studied. Age, gender, BMI, duration of diabetes, chronic complications, HbA1c, lipid profile, creatinine, albumin, PA, RBP, and retinol were recorded. High and low parameter groups were compared by Mann–Whitney U and χ2 tests. Correlation between parameters was analyzed by Spearman's test. Odds of low levels were analyzed by univariate logistic regression and included in the multivariate analysis when significant. Results. 49.5%, 48.4%, and 30.1% of patients displayed serum PA, RBP, and retinol levels below normal values, respectively. A high correlation (Rho > 0.8) between PA, RBP, and retinol serum levels was found. Patients presenting low levels of any of them were predominantly women, normal-weighted, and with lower levels of triglycerides and serum creatinine. No differences in age, macrovascular complications, duration of diabetes, or HbA1c values were observed when comparing low and normal parameter groups. Conclusion. Low serum levels of PA, RBP, and retinol are frequent in T1D adult patients. This alteration is influenced by female sex and serum creatinine and triglyceride levels. PMID:28018921

  11. Nuclear levels and patterns of histone H3 modification and HP1 proteins after inhibition of histone deacetylases.

    PubMed

    Bártová, Eva; Pacherník, Jirí; Harnicarová, Andrea; Kovarík, Ales; Kovaríková, Martina; Hofmanová, Jirina; Skalníková, Magdalena; Kozubek, Michal; Kozubek, Stanislav

    2005-11-01

    The effects of the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate (NaBt) were studied in A549, HT29 and FHC human cell lines. Global histone hyperacetylation, leading to decondensation of interphase chromatin, was characterized by an increase in H3(K9) and H3(K4) dimethylation and H3(K9) acetylation. The levels of all isoforms of heterochromatin protein, HP1, were reduced after HDAC inhibition. The observed changes in the protein levels were accompanied by changes in their interphase patterns. In control cells, H3(K9) acetylation and H3(K4) dimethylation were substantially reduced to a thin layer at the nuclear periphery, whereas TSA and NaBt caused the peripheral regions to become intensely acetylated at H3(K9) and dimethylated at H3(K4). The dispersed pattern of H3(K9) dimethylation was stable even at the nuclear periphery of HDACi-treated cells. After TSA and NaBt treatment, the HP1 proteins were repositioned more internally in the nucleus, being closely associated with interchromatin compartments, while centromeric heterochromatin was relocated closer to the nuclear periphery. These findings strongly suggest dissociation of HP1 proteins from peripherally located centromeres in a hyperacetylated and H3(K4) dimethylated environment. We conclude that inhibition of histone deacetylases caused dynamic reorganization of chromatin in parallel with changes in its epigenetic modifications.

  12. High-level expression, purification and production of the fungal immunomodulatory protein-gts in baculovirus-infected insect larva.

    PubMed

    Wu, Tzong-Yuan; Chen, Hsin-An; Li, Feng-Yin; Lin, Ching-Ting; Wu, Chi-Ming; Hsieh, Feng-Chia; Tzen, Jason Tze-Cheng; Hsieh, Sheng-Kuo; Ko, Jiunn-Liang; Jinn, Tzyy-Rong

    2013-02-01

    Fip-gts, a fungal immunomodulatory protein (Fip) isolated from Ganoderma tsugae (gts), has been reported to possess therapeutic effects in the treatment of cancer and autoimmune disease. To cost-effectively produce Fip-gts and bypass the bottleneck involved in its time-consuming purification from G. tsugae, in this study, we incorporated the SP(bbx) secretion signal into recombinant baculovirus for expressing glycosylated and bioactive rFip-gts in baculovirus-infected insect cells and Trichoplusia ni larva. This is the first study to employ the aerosol infecting T. ni larva with recombinant baculovirus for economical and high-level production of foreign proteins. In this study, one purification could yield 10 mg of rFip-gts protein merely from ∼100 infected T. ni larvae by aerosol inoculation, corresponding to 5 L (5 × 10⁹ cells) of the infected Sf21 culture. In addition, the rFip-gts purified from T. ni larvae could induce the expression of interleukin-2 in murine splenocytes with an immunoresponsive level similar to that induced by LZ-8 (a known potent immunomodulatory protein purified from Ling zhi, Ganoderma lucidum). Thus, our results demonstrated that the larva-based baculovirus expression system can successfully express rFip-gts with the assembling capability required for maintaining immunomodulatory and anticancer activity. Our approach will open a new avenue for the production of rFip-gts and facilitate the immunoregulatory activity of rFip-gts available in the future.

  13. The level of major proteins and minerals in the blood serum of chickens fed diets with pure cellulose.

    PubMed

    Bogusławska-Tryk, Monika; Szymeczko, Roman; Piotrowska, Anna

    2012-01-01

    The aim of the research was to determine the concentration of total protein and its fractions as well as the concentration of selected mineral components in the blood serum of male broiler chickens Cobb 500 fed diets with different cellulose content. Blood samples were collected for examination from the birds' pterygoid canal veins on their 42 day of age. There was no influence of cellulose preparation on the content of total protein and its fractions: albumins, alfa1-, alfa2-, beta-, gamma-globulins, albumin to globulin ratio, inorganic phosphorus, sodium, potassium, chloride and iron concentrations in blood serum. The highest calcium concentration (P < 0.05) was detected in the blood serum of males fed a diet with the highest cellulose content (0.75-0.95%). Simultaneously, a tendency of increased calcium content was observed along with an increased amount of pure cellulose in diets. The lowest magnesium level (P < 0.05) was observed in the blood of birds fed diets with the lowest amount of cellulose (0.25-0.45%). The magnesium level in the blood of birds fed a diet with higher amounts of cellulose did not considerably differ from the control group. Results from the current study suggest that introduction of a limited amount of pure cellulose into the diet of broiler chickens does not affect total protein concentration and protein fractions but can influence the mineral content in the blood serum.

  14. The Orphan G Protein-coupled Receptor Gpr175 (Tpra40) Enhances Hedgehog Signaling by Modulating cAMP Levels*

    PubMed Central

    Singh, Jaskirat; Wen, Xiaohui; Scales, Suzie J.

    2015-01-01

    The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as Tpra1 or Tpra40: transmembrane protein, adipocyte associated 1 or of 40 kDa), which also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway. PMID:26451044

  15. Levels of transaminases, alkaline phosphatase, and protein in tissues of Clarias gariepienus fingerlings exposed to sublethal concentrations of cadmium chloride.

    PubMed

    Velmurugan, Babu; Selvanayagam, Mariadoss; Cengiz, Elif I; Uysal, Ersin

    2008-12-01

    The freshwater fish, Clarias gariepienus fingerlings, were exposed to sublethal concentrations (1.7 and 3.4 mg/L) of cadmium chloride for 12 days. Aspartate aminotransferase (AAT), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total protein levels were assayed in the gill, brain, and muscle of the fish at regular intervals of 6 and 12 days. The activities of AAT, ALT, and ALP of the treated fishes increased significantly in all the tissues compared with the control fish. Protein level in all the tissues showed a significant decrease in comparison to unexposed controls throughout the experimental periods. These results revealed that cadmium chloride effects the intermediary metabolism of C. gariepienus fingerlings and that the assayed enzymes can work as good biomarkers of contamination.

  16. Salvianolic acid A positively regulates PTEN protein level and inhibits growth of A549 lung cancer cells

    PubMed Central

    BI, LEI; CHEN, JIANPING; YUAN, XIAOJING; JIANG, ZEQUN; CHEN, WEIPING

    2013-01-01

    Salvianolic acid A (Sal A) is an effective compound extracted from Salvia miltiorrhiza which has been used in the treatment of various diseases. Preliminary data indicate that Sal A treatment has a specific anti-lung cancer effect. However, the manner in which Sal A regulates cancer growth remains unknown. In this study, the A549 lung cancer cell line and its response to Sal A treatment was examined. Results showed that Sal A treatment significantly decreased A549 cell growth, promoted partial apoptosis and increased mitochondrial membrane permeability. Western blot analysis showed that Sal A upregulated the phosphatase and tensin homolog (PTEN) protein level, while consistently downregulating Akt phosphorylation. These results indicate that Sal A negatively mediates A549 lung cancer cell line growth or apoptosis, most likely by positively regulating PTEN protein level. PMID:24648921

  17. Codon-level information improves predictions of inter-residue contacts in proteins by correlated mutation analysis

    PubMed Central

    Jacob, Etai; Unger, Ron; Horovitz, Amnon

    2015-01-01

    Methods for analysing correlated mutations in proteins are becoming an increasingly powerful tool for predicting contacts within and between proteins. Nevertheless, limitations remain due to the requirement for large multiple sequence alignments (MSA) and the fact that, in general, only the relatively small number of top-ranking predictions are reliable. To date, methods for analysing correlated mutations have relied exclusively on amino acid MSAs as inputs. Here, we describe a new approach for analysing correlated mutations that is based on combined analysis of amino acid and codon MSAs. We show that a direct contact is more likely to be present when the correlation between the positions is strong at the amino acid level but weak at the codon level. The performance of different methods for analysing correlated mutations in predicting contacts is shown to be enhanced significantly when amino acid and codon data are combined. DOI: http://dx.doi.org/10.7554/eLife.08932.001 PMID:26371555

  18. Effects of aerobic exercise intervention on serum cartilage oligomeric matrix protein levels and lymphocyte dna damage in obese elderly females

    PubMed Central

    Cho, Su Youn; Roh, Hee Tae

    2016-01-01

    [Purpose] The aim of the reported research was to investigate the effects of regular aerobic exercise on cartilage oligomeric matrix protein and oxidative DNA damage in obese, elderly females. [Subjects and Methods] Sixteen class I obese, elderly females, according to World Health Organization criteria, were randomly and equally assigned to a control group (n=8) or an exercise group (n=8). The exercise group participated in exercise sessions of 60 minutes per day, 3 days per week, for a period of 8 weeks. [Results] After aerobic exercise intervention, weight, body mass index, body fat, waist circumference, and DNA damage (Tail moment) were significantly decreased, compared with baseline values. In contrast, serum cartilage oligomeric matrix protein levels were not significantly different among any groups or time-points. [Conclusion] Regular aerobic exercise may be effective for reducing obesity-induced high DNA damage levels in obese females, without causing the deformation or degradation of lower extremity articular cartilage. PMID:27390441

  19. Diet-Induced Thermogenesis and Expression Levels of Thyroid Hormone Target Genes and Their Products in Rats Differ between Meat Proteins.

    PubMed

    Ezoe, Misako; Wakamatsu, Jun-Ichi; Takahata, Yoshihisa; Hasegawa, Takanori; Morimatsu, Fumiki; Nishimura, Takanori

    2016-01-01

    We compared the effects of purified meat proteins on postprandial thermogenesis and on the secretion of and responsiveness to thyroid hormones (THs) in rats. Body temperatures at 2 h after feeding were significantly higher in the chicken and mutton protein groups than in the other groups, and these proteins seem to have a strong thermogenic effect. There were no significant differences in plasma TH concentrations among the groups, but levels of TH-responsive Spot 14 protein in the liver and brown adipose tissue were significantly higher in the chicken and mutton protein groups than in the other groups. Levels of malic enzyme 1 protein in the liver and brown adipose tissue were significantly higher in the chicken protein group than in the other groups except for the mutton protein group. Furthermore, levels of uncoupling protein 1 were higher in the chicken and mutton protein groups than in the other groups. The results suggest that the difference in postprandial thermogenesis of meat is strongly dependent on meat proteins; chicken and mutton proteins are strong promoters of postprandial thermogenesis, and THs may contribute to this effect. Since strong postprandial thermogenesis and high expression levels of TH target genes and their products were not observed in the amino acid group, chicken and mutton proteins or their digested peptides might contribute to these effects.

  20. Regulation of promyelocytic leukemia (PML) protein levels and cell morphology by bovine herpesvirus 1 infected cell protein 0 (bICP0) and mutant bICP0 proteins that do not localize to the nucleus.

    PubMed

    Gaudreault, Natasha; Jones, Clinton

    2011-03-01

    BHV-1 is an important pathogen of cattle. The infected cell protein 0 (bICP0) encoded by BHV-1 is an important regulatory protein because it is constitutively expressed and can activate all viral promoters. The mechanism by which bICP0 activates viral promoters is not well understood because bICP0 does not appear to be a sequence specific binding protein. A C(3)HC(4) zinc RING (really interesting novel gene) motif at the N-terminus of bICP0 has E3 ubiquitin ligase activity, which is important for activating viral gene expression and inhibiting interferon dependent transcription. Like other alpha-herpesvirinae ICP0 homologues, bICP0 is associated with promyelocytic leukemia (PML) protein-containing nuclear domains. During productive infection of cultured cells, BHV-1 induces degradation of the PML protein, which correlates with efficient productive infection. In this study, we demonstrated that a plasmid expressing bICP0 reduces steady state levels of the PML protein, and the C(3)HC(4) zinc RING finger is important for PML degradation. Surprisingly, bICP0 mutants with an intact C(3)HC(4) zinc RING finger that lack a nuclear localization signal also reduces steady PML protein levels. In addition, mutant bICP0 proteins that primarily localize to the cytoplasm induced morphological changes in transfected cells. During productive infection, bICP0 was detected in the cytoplasm of low-passage bovine kidney, but not established bovine kidney cells. These studies demonstrated that bICP0, even when not able to efficiently localize to the nucleus, was able to induce degradation of the PML protein and alter the morphology of transfected cells.

  1. Identifying long-term memory B-cells in vaccinated children despite waning antibody levels specific for Bordetella pertussis proteins.

    PubMed

    Hendrikx, Lotte H; Oztürk, Kemal; de Rond, Lia G H; Veenhoven, Reinier H; Sanders, Elisabeth A M; Berbers, Guy A M; Buisman, Anne-Marie

    2011-02-04

    Whooping cough is a respiratory disease caused by Bordetella pertussis. Since the 1950s in developed countries pertussis vaccinations are included in the national immunization program. However, antibody levels rapidly wane after both whole cell and acellular pertussis vaccination. Therefore protection against pertussis may depend largely on long-term B- and T-cell immunities. We investigated long-term pertussis-specific memory B-cell responses in children who were primed at infant age with the Dutch wP-vaccine (ISRCTN65428640). Purified B-cells were characterized by FACS-analysis and after polyclonal stimulation memory B-cells were detected by ELISPOT-assays specific for pertussis toxin, filamentous haemagglutinin, pertactin and tetanus. In addition, plasma IgG levels directed to the same antigens were measured by a fluorescent bead-based multiplex immunoassay. Two and 3 years after wP priming as well as 2 and 5 years after the aP booster at the age of 4, low plasma IgG levels to the pertussis proteins were found. At the same time, however pertussis protein-specific memory B-cells could be detected and their number increased with age. The number of tetanus-specific memory B-cells was similar in all age groups, whereas IgG-tetanus levels were high 2 years after tetanus booster compared to pre- and 5 years post-booster levels. This study shows the presence of long-term pertussis protein-specific memory B-cells in children despite waning antibody levels after vaccination, which suggests that memory B-cells in addition to antibodies may contribute to protection against pertussis.

  2. [Alterations in heat shock protein 70 kDa levels in human neutrophils under the heat shock conditions].

    PubMed

    Boĭko, A A; Vetchinin, S S; Sapozhnikov, A M; Kovalenko, E I

    2014-01-01

    Intracellular content of heat shock proteins of 70 kDa family (HSP70) possessing chaperone and cytoprotective functions depends on specialization and functional activity of the cells. The aim of this study was to analyze the dynamics of constitutive and inducible HSP70 levels evoked by heat shock in human neutrophils, the short-lived fraction of white blood cells providing non-specific defense against bacterial pathogens. Biphasic dynamics of the intracellular HSP70 level with an increase and following decrease in 15-30 min after the heat shock was revealed by flow cytometry. This dynamics was similar for constitutive and inducible forms of HSP70. Pre-incubation of neutrophils with cycloheximide, the inhibitor of protein synthesis, did not change the intracellular HSP70 dynamics registered by flow cytometry indicating that the increased HSP70 level detected immediately after the heat shock was not mediated by de novo protein synthesis. It was confirmed by Western blotting analysis of HSP70 intracellular content. It was suggested that the elevated HSP70 level was related to conformational HSP70 molecule changes and to increased availability of HSP70 epitopes for antibody binding. Using a panel of antibodies specific to the N-terminal ATP-binding or C-terminal substrate-binding domains of HSP70 it has been demonstrated by cell immunofluorescence and flow cytometry methods that the heat shock-associated increase of the intracellular HSP70 level was mediated by HSP70 interaction with antibodies recognizing HSP70 substrate-binding domain. It was demonstrated that the decrease of intracellular HSP70 level after heat treatment could be connected with a release of both inducible and constitutive HSP70 into extracellular space. Our data suggest that stress-induced release of HSP70 from neutrophils is regulated by ABC-transporters.

  3. Gallium nitrate increases type I collagen and fibronectin mRNA and collagen protein levels in bone and fibroblast cells.

    PubMed

    Bockman, R S; Guidon, P T; Pan, L C; Salvatori, R; Kawaguchi, A

    1993-08-01

    Gallium is a Group IIIa transitional element with therapeutic efficacy in the treatment of metabolic bone disorders. Previously described antiresorptive effects of gallium on osteoclasts are not sufficient to account for the full range of effects of gallium on bone structure and metabolism. We have recently shown that gallium nitrate inhibits osteocalcin gene expression and the synthesis of osteocalcin protein, an osteoblast-specific bone matrix protein that is thought to serve as a signal to trigger osteoclastic resorption. Here we present evidence for an additional mechanism by which gallium may function to augment bone mass by altering matrix protein synthesis by osteoblastic and fibroblastic cells. Rat calvarial explants exposed to gallium nitrate for 48 h showed increased incorporation of 3H-proline into hydroxyproline and collagenase digestible protein. In addition, gallium treatment increased steady-state mRNA levels for fibronectin and type I procollagen chains in primary rat calvarial osteoblast-enriched cultures, the ROS 17/2.8 osteoblastic osteosarcoma line, and nontransformed human dermal fibroblasts. These findings suggest that the exposure of mesenchymally-derived cells to gallium results in an altered pattern of matrix protein synthesis that would favor increased bone formation.

  4. Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein.

    PubMed Central

    Munemitsu, S; Albert, I; Souza, B; Rubinfeld, B; Polakis, P

    1995-01-01

    The APC tumor-suppressor protein associates with beta-catenin, a cell adhesion protein that is upregulated by the WNT1 oncogene. We examined the effects of exogenous APC expression on the distribution and amount of beta-catenin in a colorectal cancer cell containing only mutant APC. Expression of wild-type APC caused a pronounced reduction in total beta-catenin levels by eliminating an excessive supply of cytoplasmic beta-catenin indigenous to the SW480 colorectal cancer cell line. This reduction was due to an enhanced rate of beta-catenin protein degradation. Truncated mutant APC proteins, characteristic of those associated with cancer, lacked this activity. Mutational analysis revealed that the central region of the APC protein, which is typically deleted or severely truncated in tumors, was responsible for the down-regulation of beta-catenin. These results suggest that the tumor-suppressor activity of mutant APC may be compromised due to a defect in its ability to regulate beta-catenin. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7708772

  5. Effects of dietary protein levels for gestating gilts on reproductive performance, blood metabolites and milk composition.

    PubMed

    Jang, Y D; Jang, S K; Kim, D H; Oh, H K; Kim, Y Y

    2014-01-01

    This experiment was conducted to evaluate the effects of dietary CP levels in gestation under equal lysine content on reproductive performance, blood metabolites and milk composition of gilts. A total of 25 gilts (F1, Yorkshire×Landrace) were allotted to 4 dietary treatments at breeding in a completely randomized design, and fed 1 of 4 experimental diets containing different CP levels (11%, 13%, 15%, or 17%) at 2.0 kg/d throughout the gestation. Body weight of gilts at 24 h postpartum tended to increase linearly (p = 0.09) as dietary CP level increased. In lactation, backfat thickness, ADFI, litter size and weaning to estrus interval (WEI) did not differ among dietary treatments. There were linear increases in litter and piglet weight at 21 d of lactation (p<0.05) and weight gain of litter (p<0.01) and piglet (p<0.05) throughout the lactation as dietary CP level increased. Plasma urea nitrogen levels of gilts in gestation and at 24 h postpartum were linearly elevated as dietary CP level increased (p<0.05). Free fatty acid (FFA) levels in plasma of gestating gilts increased as dietary CP level increased up to 15%, and then decreased with quadratic effects (15 d, p<0.01; 90 d, p<0.05), and a quadratic trend (70 d, p = 0.06). There were no differences in plasma FFA, glucose levels and milk composition in lactation. These results indicate that increasing dietary CP level under equal lysine content in gestation increases BW of gilts and litter performance but does not affect litter size and milk composition. Feeding over 13% CP diet for gestating gilts could be recommended to improve litter growth.

  6. Accelerating atomic-level protein simulations by flat-histogram techniques

    NASA Astrophysics Data System (ADS)

    Jónsson, Sigurður Ć.; Mohanty, Sandipan; Irbäck, Anders

    2011-09-01

    Flat-histogram techniques provide a powerful approach to the simulation of first-order-like phase transitions and are potentially very useful for protein studies. Here, we test this approach by implicit solvent all-atom Monte Carlo (MC) simulations of peptide aggregation, for a 7-residue fragment (GIIFNEQ) of the Cu/Zn superoxide dismutase 1 protein (SOD1). In simulations with 8 chains, we observe two distinct aggregated/non-aggregated phases. At the midpoint temperature, these phases coexist, separated by a free-energy barrier of height 2.7 kBT. We show that this system can be successfully studied by carefully implemented flat-histogram techniques. The frequency of barrier crossing, which is low in conventional canonical simulations, can be increased by turning to a two-step procedure based on the Wang-Landau and multicanonical algorithms.

  7. Mitochondrial localization of the low level p53 protein in proliferative cells

    SciTech Connect

    Ferecatu, Ioana; Bergeaud, Marie; Rodriguez-Enfedaque, Aida; Le Floch, Nathalie; Oliver, Lisa; Rincheval, Vincent; Renaud, Flore; Vallette, Francois M.; Mignotte, Bernard; Vayssiere, Jean-Luc

    2009-10-02

    p53 protein plays a central role in suppressing tumorigenesis by inducing cell cycle arrest or apoptosis through transcription-dependent and -independent mechanisms. Emerging publications suggest that following stress, a fraction of p53 translocates to mitochondria to induce cytochrome c release and apoptosis. However, the localization of p53 under unstressed conditions remains largely unexplored. Here we show that p53 is localized at mitochondria in absence of apoptotic stimuli, when cells are proliferating, localization observed in various cell types (rodent and human). This is also supported by acellular assays in which p53 bind strongly to mitochondria isolated from rat liver. Furthermore, the mitochondria subfractionation study and the alkaline treatment of the mitochondrial p53 revealed that the majority of mitochondrial p53 is present in the membranous compartments. Finally, we identified VDAC, a protein of the mitochondrial outer-membrane, as a putative partner of p53 in unstressed/proliferative cells.

  8. BDNF and NT-4 differentiate two pathways in the modulation of neuropeptide protein levels in postnatal hippocampal interneurons.

    PubMed

    Marty, S; Onténiente, B

    1999-05-01

    Neuropeptide protein levels in hippocampal interneurons exhibit a considerable maturation in postnatal animals. This study characterizes the role of neuronal activity in determining neuropeptide protein levels in postnatal hippocampal interneurons, and the involvement of neurotrophins. In hippocampal slices from 7-day-old rats cultured for 2 weeks, treatment with the gamma-aminobutyric acidA (GABAA) receptor antagonist bicuculline increased the staining intensity and the number of neurons immunoreactive for neuropeptide Y (NPY). An opposite effect was observed when non-N-methyl-d-aspartate (non-NMDA) excitatory transmission was blocked. The effects of either treatment were reversed after return to control medium. These findings were similar to those previously obtained on the effects of activity on somatostatin immunostaining. Blockade of endogenous tyrosine kinase neurotrophin receptors using K252a prevented the effects of bicuculline on NPY- and somatostatin-immunoreactive neurons. Application of exogenous neurotrophin-3 (NT-3) increased NPY and somatostatin protein levels in long-term but not short-term cultures, while nerve growth factor (NGF) had no effect. In contrast, brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4) did not affect equally NPY and somatostatin immunoreactivity: they mimicked the effects of bicuculline treatment on NPY-immunoreactive neurons, but exerted no conspicuous effect on somatostatin immunostaining. These results indicate that although neuronal activity plays a major role in determining neuropeptide protein levels in postnatal hippocampal interneurons, its effects on different neuropeptides might be exerted through different mechanisms, with or without the mediation of BDNF or NT-4.

  9. Enhanced Priming of Adaptive Immunity by Mycobacterium smegmatis Mutants with High-Level Protein Secretion

    PubMed Central

    Taylor, Natalie; Bahunde, Faith; Thompson, Afton; Yu, Jae-Sung; Jacobs, William R.; Letvin, Norm L.; Haynes, Barton F.

    2012-01-01

    Mycobacteria have features that make them attractive as potential vaccine vectors. The nonpathogenic and rapidly growing Mycobacterium smegmatis can express both Mycobacterium tuberculosis antigens and heterologous antigens from other pathogens, and it has been used as a viable vector for the development of live vaccines. In order to further improve antigen-specific immunogenicity of M. smegmatis, we screened a random transposon mutant library for mutants displaying enhanced efficiency of protein secretion (“high secretors”) and isolated 61 mutants showing enhanced endogenic and transgenic protein secretion. Sequence analysis identified a total of 54 genes involved in optimal secretion of insert proteins, as well as multiple independent transposon insertions localized within the same genomic loci and operons. The majority of transposon insertions occurred in genes that have no known protein secretion function. These transposon mutants were shown to prime antigen-specific CD8+ T cell responses better than the parental strain. Specifically, upon introducing the simian immunodeficiency virus (SIV) gag gene into these transposon mutant strains, we observed that they primed SIV Gag-specific CD8+ T cell responses significantly better than the control prime immunization in a heterologous prime/boost regimen. Our results reveal a dependence on bacterial secretion of mycobacterial and foreign antigens for the induction of antigen-specific CD8+ T cells in vivo. The data also suggest that these M. smegmatis transposon mutants could be used as novel live attenuated vaccine strains to express foreign antigens, such as those of human immunodeficiency virus type 1 (HIV-1), and induce strong antigen-specific T cell responses. PMID:22787192

  10. Influence of level of dietary protein or energy on effects of ractopamine in finishing swine.

    PubMed

    Mitchell, A D; Solomon, M B; Steele, N C

    1991-11-01

    The effect of ractopamine, a beta-adrenergic agonist, on growth, nutrient utilization, and carcass composition was studied in pigs fed either 18% CP, 12% CP, or 18% CP restricted (RES = 67% of ad libitum) diets. The 18 and 12% CP diets provided 3.52 and 3.68 Mcal of DE/kg, respectively. All pigs were fed a low-protein (12% CP) diet during pretreatment growth from 15 to 60 kg. Ractopamine at 20 or 30 ppm (30 ppm for RES pigs) in the diet was fed from 60 kg live BW until slaughter at 105 kg (9 pigs/treatment). No ractopamine treatment effect (P greater than .05) was observed for either daily gain or gain/feed, although gain/feed was improved by 8% in both of the ad libitum groups. Ractopamine treatment resulted (P less than .01) in an overall reduction of carcass lipid by 8%, an increase of carcass protein by 5%, and a 21% improvement in the efficiency of protein utilization; the greatest changes occurred in the pigs fed the 12% CP diet (-17%, +11%, and +32%, respectively). The ad libitum daily feed intake was 15% less for pigs fed the 12% CP diet than for those fed the 18% CP diet (P less than .01), and there was a 10% reduction in intake of both diets with the addition of ractopamine (P less than .05). Both carcass lipid and protein deposition seemed to be closely related to energy intake (P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Changes in plasma protein levels as an early indication of a bloodstream infection.

    PubMed

    Kuusela, Pentti; Saraswat, Mayank; Joenväärä, Sakari; Kaartinen, Johanna; Järvinen, Asko; Renkonen, Risto

    2017-01-01

    Blood culture is the primary diagnostic test performed in a suspicion of bloodstream infection to detect the presence of microorganisms and direct the treatment. However, blood culture is slow and time consuming method to detect blood stream infections or separate septic and/or bacteremic patients from others with less serious febrile disease. Plasma proteomics, despite its challenges, remains an important source for early biomarkers for systemic diseases and might show changes before direct evidence from bacteria can be obtained. We have performed a plasma proteomic analysis, simultaneously at the time of blood culture sampling from ten blood culture positive and ten blood culture negative patients, and quantified 172 proteins with two or more unique peptides. Principal components analysis, Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) and ROC curve analysis were performed to select protein(s) features which can classify the two groups of samples. We propose a number of candidates which qualify as potential biomarkers to select the blood culture positive cases from negative ones. Pathway analysis by two methods revealed complement activation, phagocytosis pathway and alterations in lipid metabolism as enriched pathways which are relevant for the condition. Data are available via ProteomeXchange with identifier PXD005022.

  12. Changes in plasma protein levels as an early indication of a bloodstream infection

    PubMed Central

    Joenväärä, Sakari; Kaartinen, Johanna; Järvinen, Asko; Renkonen, Risto

    2017-01-01

    Blood culture is the primary diagnostic test performed in a suspicion of bloodstream infection to detect the presence of microorganisms and direct the treatment. However, blood culture is slow and time consuming method to detect blood stream infections or separate septic and/or bacteremic patients from others with less serious febrile disease. Plasma proteomics, despite its challenges, remains an important source for early biomarkers for systemic diseases and might show changes before direct evidence from bacteria can be obtained. We have performed a plasma proteomic analysis, simultaneously at the time of blood culture sampling from ten blood culture positive and ten blood culture negative patients, and quantified 172 proteins with two or more unique peptides. Principal components analysis, Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) and ROC curve analysis were performed to select protein(s) features which can classify the two groups of samples. We propose a number of candidates which qualify as potential biomarkers to select the blood culture positive cases from negative ones. Pathway analysis by two methods revealed complement activation, phagocytosis pathway and alterations in lipid metabolism as enriched pathways which are relevant for the condition. Data are available via ProteomeXchange with identifier PXD005022. PMID:28235076

  13. Exposure to extremely low frequency electromagnetic fields alters the behaviour, physiology and stress protein levels of desert locusts.

    PubMed

    Wyszkowska, Joanna; Shepherd, Sebastian; Sharkh, Suleiman; Jackson, Christopher W; Newland, Philip L

    2016-11-03

    Electromagnetic fields (EMFs) are present throughout the modern world and are derived from many man-made sources including overhead transmission lines. The risks of extremely-low frequency (ELF) electromagnetic fields are particularly poorly understood especially at high field strengths as they are rarely encountered at ground level. Flying insects, however, can approach close to high field strength transmission lines prompting the question as to how these high levels of exposure affect behaviour and physiology. Here we utilise the accessible nervous system of the locust to ask how exposure to high levels of ELF EMF impact at multiple levels. We show that exposure to ELF EMFs above 4 mT leads to reduced walking. Moreover, intracellular recordings from an identified motor neuron, the fast extensor tibiae motor neuron, show increased spike latency and a broadening of its spike in exposed animals. In addition, hind leg kick force, produced by stimulating the extensor tibiae muscle, was reduced following exposure, while stress-protein levels (Hsp70) increased. Together these results suggest that ELF EMF exposure has the capacity to cause dramatic effects from behaviour to physiology and protein expression, and this study lays the foundation to explore the ecological significance of these effects in other flying insects.

  14. Exposure to extremely low frequency electromagnetic fields alters the behaviour, physiology and stress protein levels of desert locusts

    PubMed Central

    Wyszkowska, Joanna; Shepherd, Sebastian; Sharkh, Suleiman; Jackson, Christopher W.; Newland, Philip L.

    2016-01-01

    Electromagnetic fields (EMFs) are present throughout the modern world and are derived from many man-made sources including overhead transmission lines. The risks of extremely-low frequency (ELF) electromagnetic fields are particularly poorly understood especially at high field strengths as they are rarely encountered at ground level. Flying insects, however, can approach close to high field strength transmission lines prompting the question as to how these high levels of exposure affect behaviour and physiology. Here we utilise the accessible nervous system of the locust to ask how exposure to high levels of ELF EMF impact at multiple levels. We show that exposure to ELF EMFs above 4 mT leads to reduced walking. Moreover, intracellular recordings from an identified motor neuron, the fast extensor tibiae motor neuron, show increased spike latency and a broadening of its spike in exposed animals. In addition, hind leg kick force, produced by stimulating the extensor tibiae muscle, was reduced following exposure, while stress-protein levels (Hsp70) increased. Together these results suggest that ELF EMF exposure has the capacity to cause dramatic effects from behaviour to physiology and protein expression, and this study lays the foundation to explore the ecological significance of these effects in other flying insects. PMID:27808167

  15. Changes in surfactant protein A mRNA levels in a rat model of insulin-treated diabetic pregnancy.

    PubMed

    Moglia, B B; Phelps, D S

    1996-02-01

    Maternal diabetes during pregnancy is associated with increased risk of neonatal respiratory distress syndrome (RDS). Previous studies using rat models for the diabetic pregnancy have documented decreased amounts of surfactant protein mRNA in the lungs of fetuses. In this study, we measured fetal lung surfactant-associated protein A (SP-A) mRNA from diabetic rats treated with insulin by daily injection or osmotic pump. Lungs were taken from fetuses on gestational d 20, and RNA was isolated and subjected to Northern blotting and densitometry to quantify SP-A mRNA. Fetal lung SP-A mRNA from untreated diabetic pregnancies was 34 +/- 2.9% of control. Insulin treatment increased levels to 55 +/- 4.2% of control values. Fetal lung SP-A mRNA levels were affected by the timing, length, and effectiveness of insulin treatment. Although levels from all treatment groups were still less than control values, insulin treatment during the last 5 or 10 d of pregnancy resulted in a substantial increase in SP-A mRNA levels over those of from untreated diabetic pregnancies. However, fetuses from the group with insulin treatment for the entire pregnancy showed decreases in fetal SP-A mRNA levels. Although the mechanism(s) responsible for the effects of diabetes and its treatment on fetal SP-A expression remain unclear, it appears unlikely that hyperglycemia is the principal cause.

  16. Fusion protein-based biofilm fabrication composed of recombinant azurin-myoglobin for dual-level biomemory application

    NASA Astrophysics Data System (ADS)

    Lee, Taek; Chung, Yong-Ho; Yoon, Jinho; Min, Junhong; Choi, Jeong-Woo

    2014-11-01

    In the present study, a fusion protein-based biofilm composed of a recombinant azurin-myoglobin (Azu-Myo) has been developed and confirmed its original electrochemical property for dual-level biomemory device application. For this purpose, the azurin was modified with cysteine residues for direct immobilization and conjugation. Then, the recombinant azurin was conjugated with the myoglobin via a sulfo-SMCC bifunctional linker using the chemical ligation method (CLM). The SDS-PAGE and UV-vis spectroscopy were performed to examine the fusion protein conjugates. The prepared Azu-Myo fusion protein was self-assembled onto Au substrate for the biofilm fabrication. Then, the atomic force microscopy (AFM) was used to confirm the immobilization and the surface-enhanced Raman spectroscopy (SERS) was carried out to the surface analysis. Also, the cyclic voltammetry (CV) was carried out to observe an electrochemical property of fabricated biofilm. As a result, the two pair of redox potential values was obtained for dual-level biomemory device application. Then, the dual-level biomemory function was verified by the multi-potential chronoamperometry (MPCA). The results indicate a new fabrication method and material combination for advances in bioelectronic device development.

  17. Association of levels of antibodies from patients with inflammatory bowel disease with extracellular proteins of food and probiotic bacteria.

    PubMed

    Hevia, Arancha; López, Patricia; Suárez, Ana; Jacquot, Claudine; Urdaci, María C; Margolles, Abelardo; Sánchez, Borja

    2014-01-01

    Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG) developed against extracellular protein fractions produced by different food bacteria from the genera Bifidobacterium and Lactobacillus. We used a sera collection consisting of healthy individuals (HC, n = 50), Crohn's disease patients (CD, n = 37), and ulcerative colitis patients (UC, n = 15). Levels of IgA antibodies developed against a cell-wall hydrolase from Lactobacillus casei subsp. rhamnosus GG (CWH) were significantly higher in the IBD group (P < 0.002; n = 52). The specificity of our measurements was confirmed by measuring IgA antibodies developed against the CWH peptide 365-VNTSNQTAAVSAS-377. IBD patients appeared to have different immune response to food bacteria. This paper sets the basis for developing systems for early detection of IBD, based on the association of high levels of antibodies developed against extracellular proteins from food and probiotic bacteria.

  18. Association of Levels of Antibodies from Patients with Inflammatory Bowel Disease with Extracellular Proteins of Food and Probiotic Bacteria

    PubMed Central

    Hevia, Arancha; López, Patricia; Suárez, Ana; Jacquot, Claudine; Urdaci, María C.; Margolles, Abelardo; Sánchez, Borja

    2014-01-01

    Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG) developed against extracellular protein fractions produced by different food bacteria from the genera Bifidobacterium and Lactobacillus. We used a sera collection consisting of healthy individuals (HC, n = 50), Crohn's disease patients (CD, n = 37), and ulcerative colitis patients (UC, n = 15). Levels of IgA antibodies developed against a cell-wall hydrolase from Lactobacillus casei subsp. rhamnosus GG (CWH) were significantly higher in the IBD group (P < 0.002; n = 52). The specificity of our measurements was confirmed by measuring IgA antibodies developed against the CWH peptide 365-VNTSNQTAAVSAS-377. IBD patients appeared to have different immune response to food bacteria. This paper sets the basis for developing systems for early detection of IBD, based on the association of high levels of antibodies developed against extracellular proteins from food and probiotic bacteria. PMID:24991549

  19. Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed.

    PubMed

    Hood, Elizabeth E; Love, Robert; Lane, Jeff; Bray, Jeff; Clough, Richard; Pappu, Kamesh; Drees, Carol; Hood, Kendall R; Yoon, Sangwoong; Ahmad, Atta; Howard, John A

    2007-11-01

    Ethanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize (Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-beta-1,4-glucanase from Acidothermus cellulolyticus, was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei, also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall. With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.

  20. Relationship Between the Hypersensitive c-Reactive Protein (hs-CRP) Level and the Prognosis of Acute Brainstem Infarction.

    PubMed

    Zhang, Xia; Huang, Wen-Juan; Yu, Zhi-Gang

    2015-05-01

    The objective of this study is to explore the relationship between the hypersensitive c-reactive protein (hs-CRP) level and the prognosis of acute brainstem infarction. Serum levels of hs-CRP were measured in 68 patients with acute brainstem infarction 72 h after disease onset. The hs-CRP levels in the U.S. National Institutes of Health Stroke Scale (NIHSS) score group and in the modified RANKIN scale (mRS) score group were compared. The independent risk factors of brainstem infarction were analyzed using Logistic binary regression. The hs-CRP level was significantly higher in the group with NIHSS >5 compared with the one with NIHSS ≤ 5 (P = 0.004). In the group with mRS > 2, the age, smoking history, and blood glucose