Brain–blood amino acid correlates following protein restriction in murine maple syrup urine disease

PubMed Central

2014-01-01

Background Conventional therapy for patients with maple syrup urine disease (MSUD) entails restriction of protein intake to maintain acceptable levels of the branched chain amino acid, leucine (LEU), monitored in blood. However, no data exists on the correlation between brain and blood LEU with protein restriction, and whether correction in blood is reflected in brain. Methods To address this question, we fed intermediate MSUD mice diets of 19% (standard) and 6% protein, with collection of sera (SE), striata (STR), cerebellum (CE) and cortex (CTX) for quantitative amino acid analyses. Results LEU and valine (VAL) levels in all brain regions improved on average 28% when shifting from 19% to 6% protein, whereas the same improvements in SE were on average 60%. Isoleucine (ILE) in brain regions did not improve, while the SE level improved 24% with low-protein consumption. Blood-branched chain amino acids (LEU, ILE, and VAL in sera (SE)) were 362-434 μM, consistent with human values considered within control. Nonetheless, numerous amino acids in brain regions remained abnormal despite protein restriction, including glutamine (GLN), aspartate (ASP), glutamate (GLU), gamma-aminobutyric acid (GABA), asparagine (ASN), citrulline (CIT) and serine (SER). To assess the specificity of these anomalies, we piloted preliminary studies in hyperphenylalaninemic mice, modeling another large neutral aminoacidopathy. Employing an identical dietary regimen, we found remarkably consistent abnormalities in GLN, ASP, and GLU. Conclusions Our results suggest that blood amino acid analysis may be a poor surrogate for assessing the outcomes of protein restriction in the large neutral amino acidopathies, and further indicate that chronic neurotransmitter disruptions (GLU, GABA, ASP) may contribute to long-term neurocognitive dysfunction in these disorders. PMID:24886632

Brain-blood amino acid correlates following protein restriction in murine maple syrup urine disease.

PubMed

Vogel, Kara R; Arning, Erland; Wasek, Brandi L; McPherson, Sterling; Bottiglieri, Teodoro; Gibson, K Michael

2014-05-08

Conventional therapy for patients with maple syrup urine disease (MSUD) entails restriction of protein intake to maintain acceptable levels of the branched chain amino acid, leucine (LEU), monitored in blood. However, no data exists on the correlation between brain and blood LEU with protein restriction, and whether correction in blood is reflected in brain. To address this question, we fed intermediate MSUD mice diets of 19% (standard) and 6% protein, with collection of sera (SE), striata (STR), cerebellum (CE) and cortex (CTX) for quantitative amino acid analyses. LEU and valine (VAL) levels in all brain regions improved on average 28% when shifting from 19% to 6% protein, whereas the same improvements in SE were on average 60%. Isoleucine (ILE) in brain regions did not improve, while the SE level improved 24% with low-protein consumption. Blood-branched chain amino acids (LEU, ILE, and VAL in sera (SE)) were 362-434 μM, consistent with human values considered within control. Nonetheless, numerous amino acids in brain regions remained abnormal despite protein restriction, including glutamine (GLN), aspartate (ASP), glutamate (GLU), gamma-aminobutyric acid (GABA), asparagine (ASN), citrulline (CIT) and serine (SER). To assess the specificity of these anomalies, we piloted preliminary studies in hyperphenylalaninemic mice, modeling another large neutral aminoacidopathy. Employing an identical dietary regimen, we found remarkably consistent abnormalities in GLN, ASP, and GLU. Our results suggest that blood amino acid analysis may be a poor surrogate for assessing the outcomes of protein restriction in the large neutral amino acidopathies, and further indicate that chronic neurotransmitter disruptions (GLU, GABA, ASP) may contribute to long-term neurocognitive dysfunction in these disorders.

Adhesives from modified soy protein

DOEpatents

Sun, Susan [Manhattan, KS; Wang, Donghai [Manhattan, KS; Zhong, Zhikai [Manhattan, KS; Yang, Guang [Shanghai, CN

2008-08-26

The present invention provides useful adhesive compositions having similar adhesive properties to conventional UF and PPF resins. The compositions generally include a protein portion and modifying ingredient portion selected from the group consisting of carboxyl-containing compounds, aldehyde-containing compounds, epoxy group-containing compounds, and mixtures thereof. The composition is preferably prepared at a pH level at or near the isoelectric point of the protein. In other preferred forms, the adhesive composition includes a protein portion and a carboxyl-containing group portion.

Enzymatic and biochemical properties of a novel human serine dehydratase isoform.

PubMed

Ogawa, Hirofumi; Gomi, Tomoharu; Nishizawa, Mikio; Hayakawa, Yumiko; Endo, Shunro; Hayashi, Kyoko; Ochiai, Hiroshi; Takusagawa, Fusao; Pitot, Henry C; Mori, Hisashi; Sakurai, Hiroaki; Koizumi, Keiichi; Saiki, Ikuo; Oda, Hirofumi; Fujishita, Takashi; Miwa, Toshiro; Maruyama, Muneharu; Kobayashi, Masashi

2006-05-01

A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showed l-serine and l-threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their Km and Vmax constants were different in a range of two-order. Removal of Pro128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and Kd value for pyridoxal 5'-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low.

Dairy cow responses to graded levels of rapeseed and soya bean expeller supplementation on a red clover/grass silage-based diet.

PubMed

Rinne, M; Kuoppala, K; Ahvenjärvi, S; Vanhatalo, A

2015-12-01

The effects of rapeseed and soya bean expeller (SBE) supplementation on digestion and milk production responses in dairy cows were investigated in an incomplete Latin square design using five cows and four 3-week periods. The experimental diets consisted of five concentrate treatments fed at a rate of 9 kg/day: a mixture of barley and oats, which was replaced with rapeseed or SBE at two levels (CP concentration (g/kg dry matter (DM)) of 130 for the control concentrate and 180 and 230 for the two protein supplemented levels). A mixture of grass and red clover silage (1:1) was fed ad libitum and it had a CP concentration of 157 g/kg DM. Supply of nutrients to the lower tract was measured using the omasal canal sampling technique, and total digestion from total faecal collection. Protein supplementation increased omasal canal amino acid (AA) flows and plasma concentrations of AA, and was also reflected as increased milk production. However, N use efficiency (NUE) decreased with increased protein supplementation. Rapeseed expeller (RSE) tended to increase silage DM intake and elicited higher milk production responses compared with SBE and also resulted in a higher NUE. The differences between the protein supplements in nitrogen metabolism were relatively small, for example, there were no differences in the efficiency of microbial protein synthesis or omasal canal flows of nitrogenous components between them, but plasma methionine concentration was lower for soya bean-fed cows at the high CP level in particular. The lower milk protein production responses to SBE than to RSE supplementation were at least partly caused by increased silage DM and by the lower methionine supply, which may further have been amplified by the use of red clover in the basal diet. Although feed intake, diet digestion, AA supply and milk production were all consistently improved by protein supplementation, there was a simultaneous decrease in NUE. In the current study, the milk protein production increased only 9% and energy-corrected milk production by 7% when high level of protein supplementation (on average 2.9 kg DM/day) was compared with the control diet without protein supplementation showing that dairy production could be sustained at a high level even without external protein supplements, at least in the short term. The economic and environmental aspects need to be carefully evaluated when decisions about protein supplementation for dairy cows are taken.

Selenium levels in human breast carcinoma tissue are associated with a common polymorphism in the gene for SELENOP (Selenoprotein P).

PubMed

Ekoue, Dede N; Zaichick, Sofia; Valyi-Nagy, Klara; Picklo, Matthew; Lacher, Craig; Hoskins, Kent; Warso, Michael A; Bonini, Marcelo G; Diamond, Alan M

2017-01-01

Selenium supplementation of the diets of rodents has consistently been shown to suppress mammary carcinogenesis and some, albeit not all, human epidemiological studies have indicated an inverse association between selenium and breast cancer risk. In order to better understand the role selenium plays in breast cancer, 30 samples of tumor tissue were obtained from women with breast cancer and analyzed for selenium concentration, the levels of several selenium-containing proteins and the levels of the MnSOD anti-oxidant protein. Polymorphisms within the genes for these same proteins were determined from DNA isolated from the tissue samples. There was a wide range of selenium in these tissues, ranging from 24 to 854ng/gm. The selenium levels in the tissues were correlated to the genotype of the SELENOP selenium carrier protein, but not to other proteins whose levels have been reported to be responsive to selenium availability, including GPX1, SELENOF and SBP1. There was an association between a polymorphism in the gene for MnSOD and the levels of the encoded protein. These studies were the first to examine the relationship between selenium levels, genotypes and protein levels in human tissues. Furthermore, the obtained data provide evidence for the need to obtain data about the effects of selenium in breast cancer by examining samples from that particular tissue type. Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balachandran, Manasi; Giannone, Richard J.; Bemis, David A.

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins withmore » an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.« less

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

DOE PAGES

Balachandran, Manasi; Giannone, Richard J.; Bemis, David A.; ...

2017-08-31

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins withmore » an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.« less

Proteomic analysis of extracellular proteins from Aspergillus oryzae grown under submerged and solid-state culture conditions.

PubMed

Oda, Ken; Kakizono, Dararat; Yamada, Osamu; Iefuji, Haruyuki; Akita, Osamu; Iwashita, Kazuhiro

2006-05-01

Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae because of its ability to produce heterologous proteins in solid-state culture. To provide an overview of protein secretion by A. oryzae in solid-state culture, we carried out a comparative proteome analysis of extracellular proteins in solid-state and submerged (liquid) cultures. Extracellular proteins prepared from both cultures sequentially from 0 to 40 h were subjected to two-dimensional electrophoresis, and protein spots at 40 h were identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. We also attempted to identify cell wall-bound proteins of the submerged culture. We analyzed 85 spots from the solid-state culture and 110 spots from the submerged culture. We identified a total of 29 proteins, which were classified into 4 groups. Group 1 consisted of extracellular proteins specifically produced in the solid-state growth condition, such as glucoamylase B and alanyl dipeptidyl peptidase. Group 2 consisted of extracellular proteins specifically produced in the submerged condition, such as glucoamylase A (GlaA) and xylanase G2 (XynG2). Group 3 consisted of proteins produced in both conditions, such as xylanase G1. Group 4 consisted of proteins that were secreted to the medium in the solid-state growth condition but trapped in the cell wall in the submerged condition, such as alpha-amylase (TAA) and beta-glucosidase (Bgl). A Northern analysis of seven genes from the four groups suggested that the secretion of TAA and Bgl was regulated by trapping these proteins in the cell wall in submerged culture and that secretion of GlaA and XynG2 was regulated at the posttranscriptional level in the solid-state culture.

Proteomic Analysis of Extracellular Proteins from Aspergillus oryzae Grown under Submerged and Solid-State Culture Conditions

PubMed Central

Oda, Ken; Kakizono, Dararat; Yamada, Osamu; Iefuji, Haruyuki; Akita, Osamu; Iwashita, Kazuhiro

2006-01-01

Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae because of its ability to produce heterologous proteins in solid-state culture. To provide an overview of protein secretion by A. oryzae in solid-state culture, we carried out a comparative proteome analysis of extracellular proteins in solid-state and submerged (liquid) cultures. Extracellular proteins prepared from both cultures sequentially from 0 to 40 h were subjected to two-dimensional electrophoresis, and protein spots at 40 h were identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. We also attempted to identify cell wall-bound proteins of the submerged culture. We analyzed 85 spots from the solid-state culture and 110 spots from the submerged culture. We identified a total of 29 proteins, which were classified into 4 groups. Group 1 consisted of extracellular proteins specifically produced in the solid-state growth condition, such as glucoamylase B and alanyl dipeptidyl peptidase. Group 2 consisted of extracellular proteins specifically produced in the submerged condition, such as glucoamylase A (GlaA) and xylanase G2 (XynG2). Group 3 consisted of proteins produced in both conditions, such as xylanase G1. Group 4 consisted of proteins that were secreted to the medium in the solid-state growth condition but trapped in the cell wall in the submerged condition, such as α-amylase (TAA) and β-glucosidase (Bgl). A Northern analysis of seven genes from the four groups suggested that the secretion of TAA and Bgl was regulated by trapping these proteins in the cell wall in submerged culture and that secretion of GlaA and XynG2 was regulated at the posttranscriptional level in the solid-state culture. PMID:16672490

Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, K.; Chubb, C.; Huberman, E.

High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteinsmore » were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.« less

Effects of a high-protein/low carbohydrate versus a standard hypocaloric diet on adipocytokine levels and insulin resistance in obese patients along 9 months.

PubMed

de Luis, Daniel Antonio; Izaola, Olatz; Aller, Rocio; de la Fuente, Beatriz; Bachiller, Rosario; Romero, Enrique

2015-01-01

Recent dietary trials and observational studies have focused on the effects of diet on health outcomes such as improvement in levels of surrogate biomarkers. The aim of our study was to examine the changes in weight, adipocytokines levels and insulin resistance after a high-protein/low carbohydrate hypocaloric diet vs. a standard hypocaloric diet during an intervention of 9 months. 331 obese subjects were randomly allocated to one of two diets for a period of 9 months. Diet HP (n=168) (high-protein hypocaloric diet) consisted in a diet of 1050 cal/day, 33% of carbohydrates, 33% of fats and 34% of proteins. Diet S (n=163) (standard protein hypocaloric diet) consisted in a diet of 1093 cal/day, 53% carbohydrates, 27%fats, and 20% proteins. With the diets HP and S, BMI, weight, fat mass, waist circumference, waist-to-hip ratio, systolic blood pressure, total cholesterol, LDL-cholesterol, insulin and HOMA decreased. The decrease at 9 months of (BMI: -2.6±1.3kg/m(2) vs. -2.1±1.2kg/m(2):p<0.05), weight (-8.4±4.2kg vs. -5.0±4.1kg: p<0.05), fat mass (-5.1±4.1kg vs. -3.4±4.2kg: p<0.05), systolic blood pressure (-5.1±7.1mmHg vs. -3.1±2.1mmHg: p<0.05), (insulin levels -4.0±4.8 UI/L vs. -2.2±2.4 UI/L; p<0.05) and HOMA (-0.8±1.0 units vs. -0.3±1.0 units; p<0.05) was higher in diet HP than Diet S. With both diets, leptin levels decreased. A high-protein/low carbohydrate hypocaloric diet shows a higher weight loss, insulin and HOMA-R decreased after 9 months than a standard hypocaloric diet. The improvement in adipokine levels was similar with both diets. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking.

PubMed

Perkins, Lydia A; Yan, Qi; Schmidt, Brigitte F; Kolodieznyi, Dmytro; Saurabh, Saumya; Larsen, Mads Breum; Watkins, Simon C; Kremer, Laura; Bruchez, Marcel P

2018-02-06

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

In Silico Estimation of Translation Efficiency in Human Cell Lines: Potential Evidence for Widespread Translational Control

PubMed Central

Stevens, Stewart G.; Brown, Chris M

2013-01-01

Recently large scale transcriptome and proteome datasets for human cells have become available. A striking finding from these studies is that the level of an mRNA typically predicts no more than 40% of the abundance of protein. This correlation represents the overall figure for all genes. We present here a bioinformatic analysis of translation efficiency – the rate at which mRNA is translated into protein. We have analysed those human datasets that include genome wide mRNA and protein levels determined in the same study. The analysis comprises five distinct human cell lines that together provide comparable data for 8,170 genes. For each gene we have used levels of mRNA and protein combined with protein stability data from the HeLa cell line to estimate translation efficiency. This was possible for 3,990 genes in one or more cell lines and 1,807 genes in all five cell lines. Interestingly, our analysis and modelling shows that for many genes this estimated translation efficiency has considerable consistency between cell lines. Some deviations from this consistency likely result from the regulation of protein degradation. Others are likely due to known translational control mechanisms. These findings suggest it will be possible to build improved models for the interpretation of mRNA expression data. The results we present here provide a view of translation efficiency for many genes. We provide an online resource allowing the exploration of translation efficiency in genes of interest within different cell lines (http://bioanalysis.otago.ac.nz/TranslationEfficiency). PMID:23460887

Age-Related Changes in the Expression of the Circadian Clock Protein PERIOD in Drosophila Glial Cells

PubMed Central

Long, Dani M.; Giebultowicz, Jadwiga M.

2018-01-01

Circadian clocks consist of molecular negative feedback loops that coordinate physiological, neurological, and behavioral variables into “circa” 24-h rhythms. Rhythms in behavioral and other circadian outputs tend to weaken during aging, as evident in progressive disruptions of sleep-wake cycles in aging organisms. However, less is known about the molecular changes in the expression of clock genes and proteins that may lead to the weakening of circadian outputs. Western blot studies have demonstrated that the expression of the core clock protein PERIOD (PER) declines in the heads of aged Drosophila melanogaster flies. This age-related decline in PER does not occur in the central pacemaker neurons but has been demonstrated so far in retinal photoreceptors. Besides photoreceptors, clock proteins are also expressed in fly glia, which play important roles in neuronal homeostasis and are further categorized into subtypes based on morphology and function. While previous studies of mammalian glial cells have demonstrated the presence of functional clocks in astrocytes and microglia, it is not known which glial cell types in Drosophila express clock proteins and how their expression may change in aged individuals. Here, we conducted immunocytochemistry experiments to identify which glial subtypes express PER protein suggestive of functional circadian clocks. Glial cell subtypes that showed night-time accumulation and day-time absence in PER consistent with oscillations reported in the pacemaker neurons were selected to compare the level of PER protein between young and old flies. Our data demonstrate that some glial subtypes show rhythmic PER expression and the relative PER levels become dampened with advanced age. Identification of glial cell types that display age-related dampening of PER levels may help to understand the cellular changes that contribute to the loss of homeostasis in the aging brain. PMID:29375400

Molecular cloning of a C-type lectin (LvLT) from the shrimp Litopenaeus vannamei: early gene down-regulation after WSSV infection.

PubMed

Ma, Tracy Hoi Tung; Tiu, Shirley Hiu Kwan; He, Jian-Guo; Chan, Siu-Ming

2007-08-01

C-type lectin is one of the pattern-recognition proteins of the non-self innate immune system in the invertebrates. In this study, a lectin-like cDNA (LvLT) of Litopenaeus vannamei was cloned and characterized. LvLT cDNA consists of 1035 nt encoding for a protein with 345 amino acid residues. The deduced LvLT consists of two putative carbohydrate-recognition domains (CRDs) as found in most C-type lectins. The first CRD consists of an amino acid motif (QPD) for the binding of galactose and the other CRDs consist of amino acid motifs (EPN) for the binding of mannose. Except for some conserved amino acid residues, the CRD of LvLT shared an overall low amino acid sequence identity with CRDs of other lectins. Unlike other shrimp lectins, LvLT is expressed only in the hepatopancreas but not in the hemocytes as revealed by RT-PCR. When juvenile shrimp were challenged with shrimp extracts containing white spot syndrome virus (WSSV), the expression levels of LvLT decreased initially in the first 2 h and then increased to a much higher level after 4 h. The results suggest that the initial reduction in LvLT transcript level may be related to the WSSV infection in shrimp.

The origin of consistent protein structure refinement from structural averaging.

PubMed

Park, Hahnbeom; DiMaio, Frank; Baker, David

2015-06-02

Recent studies have shown that explicit solvent molecular dynamics (MD) simulation followed by structural averaging can consistently improve protein structure models. We find that improvement upon averaging is not limited to explicit water MD simulation, as consistent improvements are also observed for more efficient implicit solvent MD or Monte Carlo minimization simulations. To determine the origin of these improvements, we examine the changes in model accuracy brought about by averaging at the individual residue level. We find that the improvement in model quality from averaging results from the superposition of two effects: a dampening of deviations from the correct structure in the least well modeled regions, and a reinforcement of consistent movements towards the correct structure in better modeled regions. These observations are consistent with an energy landscape model in which the magnitude of the energy gradient toward the native structure decreases with increasing distance from the native state. Copyright © 2015 Elsevier Ltd. All rights reserved.

Dysfunction of protein kinase FA/GSK-3 alpha in lymphocytes of patients with schizophrenic disorder.

PubMed

Yang, S D; Yu, J S; Lee, T T; Yang, C C; Ni, M H; Yang, Y Y

1995-09-01

As compared to normal people, the lymphocytes of patients with schizophrenia were found to have an impairment of ATP.Mg-dependent protein phosphatase activation. More importantly, the impaired protein phosphatase activation in the lymphocytes of schizophrenic patients could be consistently and completely restored to normal by exogenous pure protein kinase FA/glycogen synthase kinase-3 alpha (kinase FA/GSK-3 alpha) (the activating factor of ATP.Mg-dependent protein phosphatase), indicating that the molecular mechanism for the impaired protein phosphatase activation in schizophrenic patients may be due to a functional loss of kinase FA/GSK-3 alpha. Immunoblotting and kinase activity analysis in an anti-kinase FA/GSK-3 alpha immunoprecipitate further demonstrate that both cellular activities and protein levels of kinase FA/GSK-3 alpha in the lymphocytes of schizophrenic patients were greatly impared as compared to normal controls. Statistical analysis revealed that the lymphocytes isolated from 37 normal people contain kinase FA/GSK-3 alpha activity in the high levels of 14.8 +/- 2.4 units/mg of cell protein, whereas the lymphocytes of 48 patients with schizophrenic disorder contain kinase FA/GSK-3 alpha activity in the low levels of 2.8 +/- 1.6 units/mg, indicating that the different levels of kinase FA/GSK-3 alpha activity between schizophrenic patients and normal people are statistically significant. Taken together, the results provide initial evidence that patients with schizophrenic disorder may have a common impairment in the protein levels and cellular activities of kinase FA/GSK-3 alpha, a multisubstrate protein kinase and a multisubstrate protein phosphatase activator in their lymphocytes.

Leucine acts in the brain to suppress food intake but does not function as a physiological signal of low dietary protein

PubMed Central

Laeger, Thomas; Reed, Scott D.; Henagan, Tara M.; Fernandez, Denise H.; Taghavi, Marzieh; Addington, Adele; Münzberg, Heike; Martin, Roy J.; Hutson, Susan M.

2014-01-01

Intracerebroventricular injections of leucine are sufficient to suppress food intake, but it remains unclear whether brain leucine signaling represents a physiological signal of protein balance. We tested whether variations in dietary and circulating levels of leucine, or all three branched-chain amino acids (BCAAs), contribute to the detection of reduced dietary protein. Of the essential amino acids (EAAs) tested, only intracerebroventricular injection of leucine (10 μg) was sufficient to suppress food intake. Isocaloric low- (9% protein energy; LP) or normal- (18% protein energy) protein diets induced a divergence in food intake, with an increased consumption of LP beginning on day 2 and persisting throughout the study (P < 0.05). Circulating BCAA levels were reduced the day after LP diet exposure, but levels subsequently increased and normalized by day 4, despite persistent hyperphagia. Brain BCAA levels as measured by microdialysis on day 2 of diet exposure were reduced in LP rats, but this effect was most prominent postprandially. Despite these diet-induced changes in BCAA levels, reducing dietary leucine or total BCAAs independently from total protein was neither necessary nor sufficient to induce hyperphagia, while chronic infusion of EAAs into the brain of LP rats failed to consistently block LP-induced hyperphagia. Collectively, these data suggest that circulating BCAAs are transiently reduced by dietary protein restriction, but variations in dietary or brain BCAAs alone do not explain the hyperphagia induced by a low-protein diet. PMID:24898843

Improving Protein Fold Recognition by Deep Learning Networks.

PubMed

Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

2015-12-04

For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl's benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold.

Individuality in nutritional preferences: a multi-level approach in field crickets.

PubMed

Han, Chang S; Jäger, Heidi Y; Dingemanse, Niels J

2016-06-30

Selection may favour individuals of the same population to differ consistently in nutritional preference, for example, because optimal diets covary with morphology or personality. We provided Southern field crickets (Gryllus bimaculatus) with two synthetic food sources (carbohydrates and proteins) and quantified repeatedly how much of each macronutrient was consumed by each individual. We then quantified (i) whether individuals were repeatable in carbohydrate and protein intake rate, (ii) whether an individual's average daily intake of carbohydrates was correlated with its average daily intake of protein, and (iii) whether short-term changes in intake of carbohydrates coincided with changes in intake of protein within individuals. Intake rates were individually repeatable for both macronutrients. However, individuals differed in their relative daily intake of carbohydrates versus proteins (i.e., 'nutritional preference'). By contrast, total consumption varied plastically as a function of body weight within individuals. Body weight-but not personality (i.e., aggression, exploration behaviour)-positively predicted nutritional preference at the individual level as large crickets repeatedly consumed a higher carbohydrate to protein ratio compared to small ones. Our finding of level-specific associations between the consumption of distinct nutritional components demonstrates the merit of applying multivariate and multi-level viewpoints to the study of nutritional preference.

Ghrelin Ameliorates Asthma by Inhibiting Endoplasmic Reticulum Stress.

PubMed

Fu, Tian; Wang, Lei; Zeng, Qingdi; Zhang, Yan; Sheng, Baowei; Han, Liping

2017-12-01

This study aimed to confirm the ameliorative effect of ghrelin on asthma and investigate its mechanism. The murine model of asthma was induced by ovalbumin (OVA) treatment and assessed by histological pathology and airway responsiveness to methacholine. The total and differential leukocytes were counted. Tumor necrosis factor α, interferon γ, interleukin-5 and interleukin-13 levels in bronchoalveolar lavage fluid were quantified by commercial kits. The protein levels in pulmonary tissues were measured by Western blot analysis. Ghrelin ameliorated the histological pathology and airway hyperresponsiveness in the OVA-induced asthmatic mouse model. Consistently, OVA-increased total and differential leukocytes and levels of tumor necrosis factor α, interferon γ, interleukin-5 and interleukin-13 in bronchoalveolar lavage fluid were significantly attenuated by ghrelin. Ghrelin prevented the increased protein levels of the endoplasmic reticulum stress markers glucose regulated protein 78 and CCAAT/enhancer binding protein homologous protein and reversed the reduced levels of p-Akt in asthmatic mice. Ghrelin might prevent endoplasmic reticulum stress activation by stimulating the Akt signaling pathway, which attenuated inflammation and ameliorated asthma in mice. Ghrelin might be a new target for asthma therapy. Copyright © 2017. Published by Elsevier Inc.

Chronic exposure to nitric oxide alters the free iron pool in endothelial cells: Role of mitochondrial respiratory complexes and heat shock proteins

PubMed Central

Ramachandran, Anup; Ceaser, Erin; Darley-Usmar, Victor M.

2004-01-01

The mechanisms of nitric oxide (NO) signaling include binding to the iron centers in soluble guanylate cyclase and cytochrome c oxidase and posttranslational modification of proteins by S-nitrosation. Low levels of NO control mitochondrial number in cells, but little is known of the impact of chronic exposure to high levels of NO on mitochondrial function in endothelial cells. The focus of this study is the interaction of NO with mitochondrial respiratory complexes in cell culture and the effect this has on iron homeostasis. We demonstrate that chronic exposure of endothelial cells to NO decreased activity and protein levels of complexes I, II, and IV, whereas citrate synthase and ATP synthase were unaffected. Inhibition of these respiratory complexes was accompanied by an increase in cellular S-nitrosothiol levels, modification of cysteines residues, and an increase in the labile iron pool. The NO-dependent increase in the free iron pool and inhibition of complex II was prevented by inhibition of mitochondrial protein synthesis, consistent with a major contribution of the organelle to iron homeostasis. In addition, inhibition of mitochondrial protein synthesis was associated with an increase in heat shock protein 60 levels, which may be an additional mechanism leading to preservation of complex II activity. PMID:14691259

The effects of high dietary protein and nitrogen levels on the preformed methyl group requirement and methionine-induced growth depression in chicks.

PubMed

Pesti, G M; Benevenga, N J; Harper, A E; Sunde, M L

1981-02-01

The chick's choline and methionine requirements are both increased by high dietary protein level. Studies were conducted to test the hypothesis that the chicks' need for preformed methyl groups is increased by high protein diets (not methionine or choline per se). Chicks fed 25% isolated soybean protein (ISP) diets responded to methionine supplementation (162 vs 110 g gained in 14 days) but not to choline (119 g vs. 110 g), while those fed 50% ISP responded to either methionine (174 g vs. 126 g) or choline (181 g vs. 126 g) supplementation. Further, neither cystine nor homocystine could replace methionine in improving the growth of chicks fed the high protein diet. In other experiments, L-methionine and betaine HCl were found to alleviate the growth depression caused by excessive levels of L-glutamic acid. Excessive levels of L-methionine had a protective effect against growth depression caused by L-glutamate and diammonium citrate, and conversely, supplementary L-serine and sodium formate were not protective against glutamic acid- or arginine-induced growth depression. The results are consistent with the hypothesis that the preformed methyl group requirement is increased by high levels of dietary protein and excessive nitrogen from a single amino acid.

Consistent prediction of GO protein localization.

PubMed

Spetale, Flavio E; Arce, Debora; Krsticevic, Flavia; Bulacio, Pilar; Tapia, Elizabeth

2018-05-17

The GO-Cellular Component (GO-CC) ontology provides a controlled vocabulary for the consistent description of the subcellular compartments or macromolecular complexes where proteins may act. Current machine learning-based methods used for the automated GO-CC annotation of proteins suffer from the inconsistency of individual GO-CC term predictions. Here, we present FGGA-CC + , a class of hierarchical graph-based classifiers for the consistent GO-CC annotation of protein coding genes at the subcellular compartment or macromolecular complex levels. Aiming to boost the accuracy of GO-CC predictions, we make use of the protein localization knowledge in the GO-Biological Process (GO-BP) annotations to boost the accuracy of GO-CC prediction. As a result, FGGA-CC + classifiers are built from annotation data in both the GO-CC and GO-BP ontologies. Due to their graph-based design, FGGA-CC + classifiers are fully interpretable and their predictions amenable to expert analysis. Promising results on protein annotation data from five model organisms were obtained. Additionally, successful validation results in the annotation of a challenging subset of tandem duplicated genes in the tomato non-model organism were accomplished. Overall, these results suggest that FGGA-CC + classifiers can indeed be useful for satisfying the huge demand of GO-CC annotation arising from ubiquitous high throughout sequencing and proteomic projects.

Age-related accumulation of the advanced glycation endproduct pentosidine in human articular cartilage aggrecan: the use of pentosidine levels as a quantitative measure of protein turnover.

PubMed

Verzijl, N; DeGroot, J; Bank, R A; Bayliss, M T; Bijlsma, J W; Lafeber, F P; Maroudas, A; TeKoppele, J M

2001-11-01

During aging, non-enzymatic glycation results in the formation and accumulation of the advanced glycation endproduct pentosidine in long-lived proteins, such as articular cartilage collagen. In the present study, we investigated whether pentosidine accumulation also occurs in cartilage aggrecan. Furthermore, pentosidine levels in aggrecan subfractions of different residence time were used to explore pentosidine levels as a quantitative measure of aggrecan turnover. In order to compare protein turnover rates, protein residence time was measured as racemization of aspartic acid. As has previously been shown for collagen, pentosidine levels increase with age in cartilage aggrecan. Consistent with the faster turnover of aggrecan compared to collagen, the rate of pentosidine accumulation was threefold lower in aggrecan than in collagen. In the subfractions of aggrecan, pentosidine levels increased with protein residence time. These pentosidine levels were used to estimate the half-life of the globular hyaluronan-binding domain of aggrecan to be 19.5 years. This value is in good agreement with the half-life of 23.5 years that was estimated based on aspartic acid racemization. In aggrecan from osteoarthritic (OA) cartilage, decreased pentosidine levels were found compared with normal cartilage, which reflects increased aggrecan turnover during the OA disease process. In conclusion, we showed that pentosidine accumulates with age in aggrecan and that pentosidine levels can be used as a measure of turnover of long-lived proteins, both during normal aging and during disease.

A Pilot Proteogenomic Study with Data Integration Identifies MCT1 and GLUT1 as Prognostic Markers in Lung Adenocarcinoma.

PubMed

Stewart, Paul A; Parapatics, Katja; Welsh, Eric A; Müller, André C; Cao, Haoyun; Fang, Bin; Koomen, John M; Eschrich, Steven A; Bennett, Keiryn L; Haura, Eric B

2015-01-01

We performed a pilot proteogenomic study to compare lung adenocarcinoma to lung squamous cell carcinoma using quantitative proteomics (6-plex TMT) combined with a customized Affymetrix GeneChip. Using MaxQuant software, we identified 51,001 unique peptides that mapped to 7,241 unique proteins and from these identified 6,373 genes with matching protein expression for further analysis. We found a minor correlation between gene expression and protein expression; both datasets were able to independently recapitulate known differences between the adenocarcinoma and squamous cell carcinoma subtypes. We found 565 proteins and 629 genes to be differentially expressed between adenocarcinoma and squamous cell carcinoma, with 113 of these consistently differentially expressed at both the gene and protein levels. We then compared our results to published adenocarcinoma versus squamous cell carcinoma proteomic data that we also processed with MaxQuant. We selected two proteins consistently overexpressed in squamous cell carcinoma in all studies, MCT1 (SLC16A1) and GLUT1 (SLC2A1), for further investigation. We found differential expression of these same proteins at the gene level in our study as well as in other public gene expression datasets. These findings combined with survival analysis of public datasets suggest that MCT1 and GLUT1 may be potential prognostic markers in adenocarcinoma and druggable targets in squamous cell carcinoma. Data are available via ProteomeXchange with identifier PXD002622.

Degradation of the human mitotic checkpoint kinase Mps1 is cell cycle-regulated by APC-cCdc20 and APC-cCdh1 ubiquitin ligases.

PubMed

Cui, Yongping; Cheng, Xiaolong; Zhang, Ce; Zhang, Yanyan; Li, Shujing; Wang, Chuangui; Guadagno, Thomas M

2010-10-22

Mps1 is a dual specificity protein kinase with key roles in regulating the spindle assembly checkpoint and chromosome-microtubule attachments. Consistent with these mitotic functions, Mps1 protein levels fluctuate during the cell cycle, peaking at early mitosis and abruptly declining during mitotic exit and progression into the G(1) phase. Although evidence in budding yeast indicates that Mps1 is targeted for degradation at anaphase by the anaphase-promoting complex (APC)-c(Cdc20) complex, little is known about the regulatory mechanisms that govern Mps1 protein levels in human cells. Here, we provide evidence for the ubiquitin ligase/proteosome pathway in regulating human Mps1 levels during late mitosis through G(1) phase. First, we showed that treatment of HEK 293T cells with the proteosome inhibitor MG132 resulted in an increase in both the polyubiquitination and the accumulation of Mps1 protein levels. Next, Mps1 was shown to co-precipitate with APC and its activators Cdc20 and Cdh1 in a cell cycle-dependent manner. Consistent with this, overexpression of Cdc20 or Cdh1 led to a marked reduction of endogenous Mps1 levels during anaphase or G(1) phase, respectively. In contrast, depletion of Cdc20 or Cdh1 by RNAi treatment both led to the stabilization of Mps1 protein during mitosis or G(1) phase, respectively. Finally, we identified a single D-box motif in human Mps1 that is required for its ubiquitination and degradation. Failure to appropriately degrade Mps1 is sufficient to trigger centrosome amplification and mitotic abnormalities in human cells. Thus, our results suggest that the sequential actions of the APC-c(Cdc20) and APC-c(Cdh1) ubiquitin ligases regulate the clearance of Mps1 levels and are critical for Mps1 functions during the cell cycle in human cells.

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.

Epigenetic alterations are involved in the overexpression of glutathione S-transferase π-1 in human colorectal cancers.

PubMed

Zhang, Rui; Kang, Kyoung Ah; Piao, Mei Jing; Kim, Ki Cheon; Zheng, Jian; Yao, Cheng Wen; Cha, Ji Won; Maeng, Young Hee; Chang, Weon Young; Moon, Pyong-Gon; Baek, Moon-Chang; Hyun, Jin Won

2014-09-01

Glutathione S-transferase π-1 (GSTP-1) is a member of the glutathione S-transferase enzyme superfamily, which catalyzes the conjugation of electrophiles to glutathione during the process of detoxification. In this study, the epigenetic alterations of GSTP-1 expression in human colorectal cancers and the underlying mechanisms were investigated. In 10 colon cancer patients, proteomic analysis revealed that expression of GSTP-1 protein was higher in tumor tissues than in paired adjacent normal tissues. Likewise, in 7 of 10 colon cancer patients, GSTP-1 protein expression was more than 1.5-fold higher in tumor tissues than in adjacent normal tissues, as determined by western blotting. Immunohistochemical data confirmed that GSTP-1 protein was expressed at higher levels in colon cancer tissues compared to normal mucosa. GSTP-1 enzyme activity was closely correlated with GSTP-1 protein expression in colon cancer patients. Consistent with this, GSTP-1 mRNA, protein and activity levels were higher in the colorectal cancer cell lines Caco-2, HCT-116, HT-29, SNU-407 and SNU-1033 compared to the normal colon cell line FHC. Methylation-specific PCR results indicated that the high levels of GSTP-1 in human colorectal cancer cell lines were likely due to the lower degree of promoter methylation in colon cancer cell lines compared to the normal colon cell line, consistent with findings in colon cancer patients. Moreover, the levels of specific activator-protein complexes and histone marks were higher in human colorectal cancer cells compared to the normal human colon cell line, whereas the repressor protein complexes exhibited the opposite pattern. Furthermore, chromatin immunoprecipitation assays demonstrated that expression levels of the transcription factors AP-1 and SP-1 were correlated with the upregulation of GSTP-1 expression in colorectal cancer cells. Finally, knockdown of GSTP-1 promoted the sensitivity of SNU-407 cells to the anticancer agent 5-fluorouracil. These data indicate that GSTP-1 may serve as a clinically useful biomarker of colon cancer and a target for anti-colon cancer drugs.

Effect of N-acetylcysteine administration on homocysteine level, oxidative damage to proteins, and levels of iron (Fe) and Fe-related proteins in lead-exposed workers.

PubMed

Kasperczyk, Sławomir; Dobrakowski, Michał; Kasperczyk, Aleksandra; Romuk, Ewa; Rykaczewska-Czerwińska, Monika; Pawlas, Natalia; Birkner, Ewa

2016-09-01

N-Acetylcysteine (NAC) could be included in protocols designed for the treatment of lead toxicity. Therefore, in this study, we decided to investigate the influence of NAC administration on homocysteine (Hcy) levels, oxidative damage to proteins, and the levels of iron (Fe), transferrin (TRF), and haptoglobin (HPG) in lead (Pb)-exposed workers. The examined population (n = 171) was composed of male employees who worked with Pb. They were randomized into four groups. Workers who were not administered any antioxidants, drugs, vitamins, or dietary supplements were classified as the reference group (n = 49). The remaining three groups consisted of workers who were treated orally with NAC at three different doses (1 × 200, 2 × 200, or 2 × 400 mg) for 12 weeks. After the treatment, blood Pb levels significantly decreased in the groups receiving NAC compared with the reference group. The protein concentration was not affected by NAC administration. In contrast, Hcy levels significantly decreased or showed a strong tendency toward lower values depending on the NAC dose. Levels of the protein carbonyl groups were significantly decreased in all of the groups receiving NAC. Conversely, glutamate dehydrogenase activity was significantly elevated in all of the groups receiving NAC, while the level of protein thiol groups was significantly elevated only in the group receiving 200 mg of NAC. Treatment with NAC did not significantly affect Fe and TRF levels, whereas HPG levels showed a tendency toward lower values. Treatment with NAC normalized the level of Hcy and decreased oxidative stress as measured by the protein carbonyl content; this effect occurred in a dose-dependent manner. Moreover, small doses of NAC elevated the levels of protein thiol groups. Therefore, NAC could be introduced as an alternative therapy for chronic Pb toxicity in humans. © The Author(s) 2015.

Reduction of blood serum cholesterol

NASA Technical Reports Server (NTRS)

Winitz, M. (Inventor)

1974-01-01

By feeding a human subject as the sole source of sustenance a defined diet wherein the carbohydrate consists substantially entirely of glucose, maltose or a polysaccharide of glucose, the blood serum cholesterol level of the human subject is substantially reduced. If 25 percent of the carbohydrate is subsequently supplied in the form of sucrose, an immediate increase from the reduced level is observed. The remainder of the defined diet normally includes a source of amino acids, such as protein or a protein hydrolysate, vitamins, minerals and a source of essential fatty acid.

NRIP/DCAF6 stabilizes the androgen receptor protein by displacing DDB2 from the CUL4A-DDB1 E3 ligase complex in prostate cancer.

PubMed

Chen, Hsin-Hsiung; Fan, Ping; Chang, Szu-Wei; Tsao, Yeou-Ping; Huang, Hsiang-Po; Chen, Show-Li

2017-03-28

Both nuclear receptor interaction protein (NRIP) and DNA damage binding protein 2 (DDB2) belong to the Cullin 4 (CUL4)-DDB1 binding protein family and are androgen receptor (AR)-interacting proteins. Here, we investigated the expression patterns of the NRIP, DDB2 and AR proteins in human prostate cancer tissues and found that the expression levels of NRIP and AR were higher, but the DDB2 level was lower, in prostate cancer tissues than in non-neoplastic controls, suggesting NRIP as a candidate tumor promoter and DDB2 as a tumor suppressor in prostate cancer. Furthermore, both NRIP and DDB2 shared the same AR binding domain; they were competitors for the AR, but not for DDB1 binding, in the AR-DDB2-DDB1-CUL4A complex. Conclusively, NRIP stabilizes the AR protein by displacing DDB2 from the AR-DDB2 complex. Consistent with our hypothesis, a specific expression pattern with high levels of NRIP and AR, together with a low level of DDB2, was found more frequently in the human prostate cancer tissues with a cribriform pattern than in non-cribriform tumors, suggesting that disruption of the balance between NRIP and DDB2 may change AR protein homeostasis and contribute to pathogenesis in certain aggressive types of prostate cancer.

NRIP/DCAF6 stabilizes the androgen receptor protein by displacing DDB2 from the CUL4A-DDB1 E3 ligase complex in prostate cancer

PubMed Central

Tsao, Yeou-Ping; Huang, Hsiang-Po; Chen, Show-Li

2017-01-01

Both nuclear receptor interaction protein (NRIP) and DNA damage binding protein 2 (DDB2) belong to the Cullin 4 (CUL4)-DDB1 binding protein family and are androgen receptor (AR)-interacting proteins. Here, we investigated the expression patterns of the NRIP, DDB2 and AR proteins in human prostate cancer tissues and found that the expression levels of NRIP and AR were higher, but the DDB2 level was lower, in prostate cancer tissues than in non-neoplastic controls, suggesting NRIP as a candidate tumor promoter and DDB2 as a tumor suppressor in prostate cancer. Furthermore, both NRIP and DDB2 shared the same AR binding domain; they were competitors for the AR, but not for DDB1 binding, in the AR-DDB2-DDB1-CUL4A complex. Conclusively, NRIP stabilizes the AR protein by displacing DDB2 from the AR-DDB2 complex. Consistent with our hypothesis, a specific expression pattern with high levels of NRIP and AR, together with a low level of DDB2, was found more frequently in the human prostate cancer tissues with a cribriform pattern than in non-cribriform tumors, suggesting that disruption of the balance between NRIP and DDB2 may change AR protein homeostasis and contribute to pathogenesis in certain aggressive types of prostate cancer. PMID:28212551

Inducible immune proteins in the dampwood termite Zootermopsis angusticollis

NASA Astrophysics Data System (ADS)

Rosengaus, Rebeca B.; Cornelisse, Tara; Guschanski, Katerina; Traniello, James F. A.

2007-01-01

Dampwood termites, Zootermopsis angusticollis (Isoptera: Termopsidae), mount an immune response to resist microbial infection. Here we report on results of a novel analysis that allowed us to electrophoretically assess changes in hemolymph proteins in the same individual before and after exposure to a pathogen. We demonstrate that contact with a sublethal concentration of the entomopathogenic fungus Metarhizium anisopliae (Deuteromycotina:Hypomycetes) induces the production of protective proteins in nymphs, pseudergates (false workers), and soldiers. Termites exposed to an immunizing dosage of fungal conidia consistently showed an enhancement of constitutive proteins (62-85 kDa) in the hemolymph as well as an induction of novel proteins (28-48 kDa) relative to preimmunization levels. No significant differences in protein banding patterns relative to baseline levels in control and naïve termites were observed. Incubating excised and eluted induced proteins produced by immunized pseudergates or immunized soldiers with conidia significantly reduced the germination of the fungus. The fungistatic effect of eluted proteins differed significantly among five colonies examined. Our results show that the upregulation of protective proteins in the hemolymph underscores the in vivo immune response we previously recorded in Z. angusticollis.

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.

Maintenance of cellular ATP level by caloric restriction correlates chronological survival of budding yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Joon-Seok; Lee, Cheol-Koo, E-mail: cklee2005@korea.ac.kr

Highlights: •CR decreases total ROS and mitochondrial superoxide during the chronological aging. •CR does not affect the levels of oxidative damage on protein and DNA. •CR contributes extension of chronological lifespan by maintenance of ATP level -- Abstract: The free radical theory of aging emphasizes cumulative oxidative damage in the genome and intracellular proteins due to reactive oxygen species (ROS), which is a major cause for aging. Caloric restriction (CR) has been known as a representative treatment that prevents aging; however, its mechanism of action remains elusive. Here, we show that CR extends the chronological lifespan (CLS) of budding yeastmore » by maintaining cellular energy levels. CR reduced the generation of total ROS and mitochondrial superoxide; however, CR did not reduce the oxidative damage in proteins and DNA. Subsequently, calorie-restricted yeast had higher mitochondrial membrane potential (MMP), and it sustained consistent ATP levels during the process of chronological aging. Our results suggest that CR extends the survival of the chronologically aged cells by improving the efficiency of energy metabolism for the maintenance of the ATP level rather than reducing the global oxidative damage of proteins and DNA.« less

The serum and glucocorticoid-regulated protein kinases (SGK) stimulate bovine herpesvirus 1 and herpes simplex virus 1 productive infection.

PubMed

Kook, Insun; Jones, Clinton

2016-08-15

Serum and glucocorticoid-regulated protein kinases (SGK) are serine/threonine protein kinases that contain a catalytic domain resembling other protein kinases: AKT/protein kinase B, protein kinase A, and protein kinase C-Zeta for example. Unlike these constitutively expressed protein kinases, SGK1 RNA and protein levels are increased by growth factors and corticosteroids. Stress can directly stimulate SGK1 levels as well as stimulate bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) productive infection and reactivation from latency suggesting SGK1 can stimulate productive infection. For the first time, we provide evidence that a specific SGK inhibitor (GSK650394) significantly reduced BoHV-1 and HSV-1 replication in cultured cells. Proteins encoded by the three BoHV-1 immediate early genes (bICP0, bICP4, and bICP22) and two late proteins (VP16 and gE) were consistently reduced by GSK650394 during early stages of productive infection. In summary, these studies suggest SGK may stimulate viral replication following stressful stimuli. Copyright © 2016 Elsevier B.V. All rights reserved.

Improving Protein Fold Recognition by Deep Learning Networks

NASA Astrophysics Data System (ADS)

Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

2015-12-01

For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl’s benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold.

Development of an electrochemical detection system for measuring DNA methylation levels using methyl CpG-binding protein and glucose dehydrogenase-fused zinc finger protein.

PubMed

Lee, Jinhee; Yoshida, Wataru; Abe, Koichi; Nakabayashi, Kazuhiko; Wakeda, Hironobu; Hata, Kenichiro; Marquette, Christophe A; Blum, Loïc J; Sode, Koji; Ikebukuro, Kazunori

2017-07-15

DNA methylation level at a certain gene region is considered as a new type of biomarker for diagnosis and its miniaturized and rapid detection system is required for diagnosis. Here we have developed a simple electrochemical detection system for DNA methylation using methyl CpG-binding domain (MBD) and a glucose dehydrogenase (GDH)-fused zinc finger protein. This analytical system consists of three steps: (1) methylated DNA collection by MBD, (2) PCR amplification of a target genomic region among collected methylated DNA, and (3) electrochemical detection of the PCR products using a GDH-fused zinc finger protein. With this system, we have successfully measured the methylation levels at the promoter region of the androgen receptor gene in 10 6 copies of genomic DNA extracted from PC3 and TSU-PR1 cancer cell lines. Since no sequence analysis or enzymatic digestion is required for this detection system, DNA methylation levels can be measured within 3h with a simple procedure. Copyright © 2016 Elsevier B.V. All rights reserved.

Control of storage-protein synthesis during seed development in pea (Pisum sativum L.).

PubMed Central

Gatehouse, J A; Evans, I M; Bown, D; Croy, R R; Boulter, D

1982-01-01

The tissue-specific syntheses of seed storage proteins in the cotyledons of developing pea (Pisum sativum L.) seeds have been demonstrated by estimates of their qualitative and quantitative accumulation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and rocket immunoelectrophoresis respectively. Vicilin-fraction proteins initially accumulated faster than legumin, but whereas legumin was accumulated throughout development, different components of the vicilin fraction had their predominant periods of synthesis at different stages of development. The translation products in vitro of polysomes isolated from cotyledons at different stages of development reflected the synthesis in vivo of storage-protein polypeptides at corresponding times. The levels of storage-protein mRNA species during development were estimated by 'Northern' hybridization using cloned complementary-DNA probes. This technique showed that the levels of legumin and vicilin (47000-Mr precursors) mRNA species increased and decreased in agreement with estimated rates of synthesis of the respective polypeptides. The relative amounts of these messages, estimated by kinetic hybridization were also consistent. Legumin mRNA was present in leaf poly(A)+ RNA at less than one-thousandth of the level in cotyledon poly(A)+ (polyadenylated) RNA, demonstrating tissue-specific expression. Evidence is presented that storage-protein mRNA species are relatively long-lived, and it is suggested that storage-protein synthesis is regulated primarily at the transcriptional level. Images Fig. 2. Fig. 3. PMID:6897609

21 CFR 866.5370 - Cohn fraction V immuno-logical test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... test system is a device that consists of or measures that fraction of plasma containing predominantly albumin (a plasma protein). This test aids in the diagnosis of diseases where albumin levels may be...

21 CFR 866.5370 - Cohn fraction V immuno-logical test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... test system is a device that consists of or measures that fraction of plasma containing predominantly albumin (a plasma protein). This test aids in the diagnosis of diseases where albumin levels may be...

21 CFR 866.5370 - Cohn fraction V immuno-logical test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... test system is a device that consists of or measures that fraction of plasma containing predominantly albumin (a plasma protein). This test aids in the diagnosis of diseases where albumin levels may be...

21 CFR 866.5370 - Cohn fraction V immuno-logical test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... test system is a device that consists of or measures that fraction of plasma containing predominantly albumin (a plasma protein). This test aids in the diagnosis of diseases where albumin levels may be...

21 CFR 866.5370 - Cohn fraction V immuno-logical test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... test system is a device that consists of or measures that fraction of plasma containing predominantly albumin (a plasma protein). This test aids in the diagnosis of diseases where albumin levels may be...

Investigating the clinical potential for 14-3-3 zeta protein to serve as a biomarker for epithelial ovarian cancer

PubMed Central

2013-01-01

Objective Recently, 14-3-3 zeta protein was identified as a potential serum biomarker of epithelial ovarian cancer (EOC). The goal of this study was to investigate the clinical potential of 14-3-3 zeta protein for monitoring EOC progression compared with CA-125 and HE4. Design Prospective follow-up study. Setting University of Pecs Medical Center Department of Obstetrics and Gynecology/Oncology (Pecs, Hungary). Population Thirteen EOC patients with advanced stage (FIGO IIb-IIIc) epithelial ovarian cancer that underwent radical surgery and received six consecutive cycles of first line chemotherapy (paclitaxel, carboplatin) in 21-day intervals. Methods Pre- and post-chemotherapy computed tomography (CT) scans were performed. Serum levels of CA-125, HE4, and 14-3-3 zeta protein were detected by enzyme-linked immunosorbent assay (ELISA) and quantitative electrochemiluminescence assay (ECLIA). Main outcome measures Serum levels of CA-125, HE4, and 14-3-3 zeta protein, as well as lesion size according to pre- and post-chemotherapy CT scans. Results Serum levels of CA-125 and HE4 were found to significantly decrease following chemotherapy, and this was consistent with the decrease in lesion size detected post-chemotherapy. In contrast, 14-3-3 zeta protein levels did not significantly differ in healthy postmenopausal patients versus EOC patients. Conclusions Determination of CA-125 and HE4 serum levels for the determination of the risk of ovarian malignancy algorithm (ROMA) represents a useful tool for the prediction of chemotherapy efficacy for EOC patients. However, levels of 14-3-3 zeta protein were not found to vary significantly as a consequence of treatment. Therefore we question if 14-3-3 zeta protein is a reliable biomarker, which correlates with the clinical behavior of EOC. PMID:24238270

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

PubMed

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

2017-06-01

Procoagulant factors promote cancer progression and metastasis. Protein C is involved in hemostasis, inflammation and signal transduction, and has a protective effect on the endothelial barrier. In mice, administration of activated protein C reduced experimental metastasis. We assessed the association between protein C and mortality in patients with three types of cancer. The study population consisted of patients with advanced prostate, non-small cell lung or pancreatic cancer, who participated in the INPACT trial (NCT00312013). The trial evaluated the addition of nadroparin to chemotherapy in patients with advanced malignancy. Patients were divided into tertiles based on protein C at baseline. The association between protein C levels and mortality was evaluated with Cox proportional hazard models. We analysed 477 patients (protein C tertiles: <97, 97-121 and ≥121%). Mean age was 65±9years; 390 (82%) were male; 191 patients (40%) had prostate cancer, 161 (34%) had lung cancer, and 125 (26%) pancreatic cancer. During a median follow-up of 10.4months, 291 patients (61%) died. Median protein C level was 107% (IQR 92-129). In the lowest tertile, 75 patients per 100 patient-years died, as compared to 60 and 54 in the middle and high tertile, respectively. Lower levels of protein C were associated with increased mortality (in tertiles: HR for trend 1.18, 95%CI 1.02-1.36, adjusted for age, sex and nadroparin use; as a continuous variable: HR 1.004, 95%CI 1.00-1.008, p=0.07). Protein C seems inversely associated with mortality in patients with advanced prostate, lung and pancreatic cancer. Further research should validate protein C as a biomarker for mortality, and explore the effects of protein C on progression of cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

Energetic costs of protein synthesis do not differ between red- and white-blooded Antarctic notothenioid fishes.

PubMed

Lewis, Johanne M; Grove, Theresa J; O'Brien, Kristin M

2015-09-01

Antarctic icefishes (Family Channichthyidae) within the suborder Notothenioidei lack the oxygen-binding protein hemoglobin (Hb), and six of the 16 species of icefishes lack myoglobin (Mb) in heart ventricle. As iron-centered proteins, Hb and Mb can promote the formation of reactive oxygen species (ROS) that damage biological macromolecules. Consistent with this, our previous studies have shown that icefishes have lower levels of oxidized proteins and lipids in oxidative muscle compared to red-blooded notothenioids. Because oxidized proteins are usually degraded by the 20S proteasome and must be resynthesized, we hypothesized that rates of protein synthesis would be lower in icefishes compared to red-blooded notothenioids, thereby reducing the energetic costs of protein synthesis and conferring a benefit to the loss of Hb and Mb. Rates of protein synthesis were quantified in hearts, and the fraction of oxygen consumption devoted to protein synthesis was measured in isolated hepatocytes and cardiomyocytes of notothenioids differing in the expression of Hb and cardiac Mb. Neither rates of protein synthesis nor the energetic costs of protein synthesis differed among species, suggesting that red-blooded species do not degrade and replace oxidatively modified proteins at a higher rate compared to icefishes but rather, persist with higher levels of oxidized proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Variations in isoflavone levels in soy foods and soy protein isolates and issues related to isoflavone databases and food labeling.

PubMed

Setchell, Kenneth D R; Cole, Sidney J

2003-07-02

The reliability of databases on the isoflavone composition of foods designed to estimate dietary intakes is contingent on the assumption that soy foods are consistent in their isoflavone content. To validate this, total and individual isoflavone compositions were determined by HPLC for two different soy protein isolates used in the commercial manufacture of soy foods over a 3-year period (n = 30/isolate) and 85 samples of 40 different brands of soy milks. Total isoflavone concentrations differed markedly between the soy protein isolates, varying by 200-300% over 3 years, whereas the protein content varied by only 3%. Total isoflavone content varied by up to 5-fold among different commercial soy milks and was not consistent between repeat purchases. Whole soybean milks had significantly higher isoflavone levels than those made from soy protein isolates (mean +/- SD, 63.6 +/- 21.9 mg/L, n = 43, vs 30.2 +/- 5.8 mg/L, n = 38, respectively, p < 0.0001), although some isolated soy protein-based milks were similar in content to "whole bean" varieties. The ratio of genistein to daidzein isoflavone forms was higher in isolated soy protein-based versus "whole bean" soy milks (2.72 +/- 0.24 vs 1.62 +/- 0.47, respectively, p < 0.0001), and the greatest variability in isoflavone content was observed among brands of whole bean soy milks. These studies illustrate large variability in the isoflavone content of isolated soy proteins used in food manufacture and in commercial soy milks and reinforce the need to accurately determine the isoflavone content of foods used in dietary intervention studies while exposing the limitations of food databases for estimating daily isoflavone intakes.

HLA-B35 Upregulates Endothelin-1 and Downregulates Endothelial Nitric Oxide Synthase via Endoplasmic Reticulum Stress Response in Endothelial Cells

PubMed Central

Lenna, Stefania; Townsend, Danyelle M.; Tan, Filemon K.; Kapanadze, Bagrat; Markiewicz, Malgorzata; Trojanowska, Maria; Scorza, Raffaella

2013-01-01

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1mRNAand protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension. PMID:20335527

HLA-B35 upregulates endothelin-1 and downregulates endothelial nitric oxide synthase via endoplasmic reticulum stress response in endothelial cells.

PubMed

Lenna, Stefania; Townsend, Danyelle M; Tan, Filemon K; Kapanadze, Bagrat; Markiewicz, Malgorzata; Trojanowska, Maria; Scorza, Raffaella

2010-05-01

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1 mRNA and protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension.

Lower glutamic acid decarboxylase 65kD mRNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia

PubMed Central

Glausier, JR; Kimoto, S; Fish, KN; Lewis, DA

2014-01-01

Background Altered GABA signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in schizophrenia and schizoaffective disorder. PFC levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67kD (GAD67) has been consistently reported to be lower in these disorders, but the status of the second GABA-synthesizing enzyme, GAD65, remains unclear. Methods GAD65 mRNA levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. GAD65 relative protein levels were quantified in a subset of subject pairs by confocal immunofluorescence microscopy. Results Mean GAD65 mRNA levels were 13.6% lower in schizoaffective disorder subjects, but did not differ in schizophrenia subjects, relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein measures within schizoaffective disorder subjects was not attributable to factors commonly comorbid with the diagnosis. Conclusions In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in subjects with schizoaffective disorder relative to subjects with schizophrenia, these findings may support an interpretation that GAD65 down-regulation provides a homeostatic response complementary to GAD67 down-regulation expression that serves to reduce inhibition in the face of lower PFC network activity. PMID:24993056

Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS

PubMed Central

Yoo, Chul; Patwa, Tasneem H.; Kreunin, Paweena; Miller, Fred R.; Huber, Christian G.; Nesvizhskii, Alexey I.; Lubman, David M.

2012-01-01

A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 μg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis. PMID:17206599

Integrative Structure Determination of Protein Assemblies by Satisfaction of Spatial Restraints

NASA Astrophysics Data System (ADS)

Alber, Frank; Chait, Brian T.; Rout, Michael P.; Sali, Andrej

To understand the cell, we need to determine the structures of macromolecular assemblies, many of which consist of tens to hundreds of components. A great variety of experimental data can be used to characterize the assemblies at several levels of resolution, from atomic structures to component configurations. To maximize completeness, resolution, accuracy, precision and efficiency of the structure determination, a computational approach is needed that can use spatial information from a variety of experimental methods. We propose such an approach, defined by its three main components: a hierarchical representation of the assembly, a scoring function consisting of spatial restraints derived from experimental data, and an optimization method that generates structures consistent with the data. We illustrate the approach by determining the configuration of the 456 proteins in the nuclear pore complex from Baker's yeast.

G protein-coupled odorant receptors: From sequence to structure.

PubMed

de March, Claire A; Kim, Soo-Kyung; Antonczak, Serge; Goddard, William A; Golebiowski, Jérôme

2015-09-01

Odorant receptors (ORs) are the largest subfamily within class A G protein-coupled receptors (GPCRs). No experimental structural data of any OR is available to date and atomic-level insights are likely to be obtained by means of molecular modeling. In this article, we critically align sequences of ORs with those GPCRs for which a structure is available. Here, an alignment consistent with available site-directed mutagenesis data on various ORs is proposed. Using this alignment, the choice of the template is deemed rather minor for identifying residues that constitute the wall of the binding cavity or those involved in G protein recognition. © 2015 The Protein Society.

The use of dissolved oxygen-controlled, fed-batch aerobic cultivation for recombinant protein subunit vaccine manufacturing.

PubMed

Farrell, Patrick; Sun, Jacob; Champagne, Paul-Philippe; Lau, Heron; Gao, Meg; Sun, Hong; Zeiser, Arno; D'Amore, Tony

2015-11-27

A simple "off-the-shelf" fed-batch approach to aerobic bacterial cultivation for recombinant protein subunit vaccine manufacturing is presented. In this approach, changes in the dissolved oxygen levels are used to adjust the nutrient feed rate (DO-stat), so that the desired dissolved oxygen level is maintained throughout cultivation. This enables high Escherichia coli cell densities and recombinant protein titers. When coupled to a kLa-matched scale-down model, process performance is shown to be consistent at the 2L, 20L, and 200L scales for two recombinant E. coli strains expressing different protein subunit vaccine candidates. Additionally, by mining historical DO-stat nutrient feeding data, a method to transition from DO-stat to a pre-determined feeding profile suitable for larger manufacturing scales without using feedback control is demonstrated at the 2L, 20L, and 200L scales. Copyright © 2015 Elsevier Ltd. All rights reserved.

Protein Requirements and Recommendations for Older People: A Review.

PubMed

Nowson, Caryl; O'Connell, Stella

2015-08-14

Declines in skeletal muscle mass and strength are major contributors to increased mortality, morbidity and reduced quality of life in older people. Recommended Dietary Allowances/Intakes have failed to adequately consider the protein requirements of the elderly with respect to function. The aim of this paper was to review definitions of optimal protein status and the evidence base for optimal dietary protein. Current recommended protein intakes for older people do not account for the compensatory loss of muscle mass that occurs on lower protein intakes. Older people have lower rates of protein synthesis and whole-body proteolysis in response to an anabolic stimulus (food or resistance exercise). Recommendations for the level of adequate dietary intake of protein for older people should be informed by evidence derived from functional outcomes. Randomized controlled trials report a clear benefit of increased dietary protein on lean mass gain and leg strength, particularly when combined with resistance exercise. There is good consistent evidence (level III-2 to IV) that consumption of 1.0 to 1.3 g/kg/day dietary protein combined with twice-weekly progressive resistance exercise reduces age-related muscle mass loss. Older people appear to require 1.0 to 1.3 g/kg/day dietary protein to optimize physical function, particularly whilst undertaking resistance exercise recommendations.

Protein Requirements and Recommendations for Older People: A Review

PubMed Central

Nowson, Caryl; O’Connell, Stella

2015-01-01

Declines in skeletal muscle mass and strength are major contributors to increased mortality, morbidity and reduced quality of life in older people. Recommended Dietary Allowances/Intakes have failed to adequately consider the protein requirements of the elderly with respect to function. The aim of this paper was to review definitions of optimal protein status and the evidence base for optimal dietary protein. Current recommended protein intakes for older people do not account for the compensatory loss of muscle mass that occurs on lower protein intakes. Older people have lower rates of protein synthesis and whole-body proteolysis in response to an anabolic stimulus (food or resistance exercise). Recommendations for the level of adequate dietary intake of protein for older people should be informed by evidence derived from functional outcomes. Randomized controlled trials report a clear benefit of increased dietary protein on lean mass gain and leg strength, particularly when combined with resistance exercise. There is good consistent evidence (level III-2 to IV) that consumption of 1.0 to 1.3 g/kg/day dietary protein combined with twice-weekly progressive resistance exercise reduces age-related muscle mass loss. Older people appear to require 1.0 to 1.3 g/kg/day dietary protein to optimize physical function, particularly whilst undertaking resistance exercise recommendations. PMID:26287239

Detection and Identification of the Vibrational Markers for the Quantification of Methionine Oxidation in Therapeutic Proteins.

PubMed

Balakrishnan, Gurusamy; Barnett, Gregory V; Kar, Sambit R; Das, Tapan K

2018-05-17

Methionine oxidation is a major degradation pathway in therapeutic proteins which can impact the structure and function of proteins as well as risk to drug product quality. Detecting Met oxidation in proteins by peptide mapping followed by liquid chromatography with mass spectrometry (LC-MS) is the industry standard but is also labor intensive and susceptible to artifacts. In this work, vibrational difference spectroscopy in combination with 18 O isotopic shift enabled us to demonstrate the application of Raman and FTIR techniques for the detection and quantification of Met oxidation in various therapeutic proteins, including mAbs, fusion proteins, and antibody drug conjugate. Vibrational markers of Met oxidation products, such as sulfoxide and sulfone, corresponding to S═O and C-S═O stretching frequencies were unequivocally identified based 18 O isotoptic shifts. The intensity of the isolated νC-S Raman band at 702 cm -1 was successfully applied to quantify the average Met oxidation level in multiple proteins. These results are further corroborated by oxidation levels measured by tryptic peptide mapping, and thus the confirmed Met oxidation levels derived from Raman and mass spectrometry are indeed consistent with each other. Thus, we demonstrate the broader application of vibrational spectroscopy to detect the subtle spectral changes associated with various chemical or physical degradation of proteins, including Met oxidation as well as higher order structural changes.

The gap junction channel protein connexin 43 is covalently modified and regulated by SUMOylation.

PubMed

Kjenseth, Ane; Fykerud, Tone A; Sirnes, Solveig; Bruun, Jarle; Yohannes, Zeremariam; Kolberg, Matthias; Omori, Yasufumi; Rivedal, Edgar; Leithe, Edward

2012-05-04

SUMOylation is a posttranslational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in specific target proteins. Most known SUMOylation target proteins are located in the nucleus, but there is increasing evidence that SUMO may also be a key determinant of many extranuclear processes. Gap junctions consist of arrays of intercellular channels that provide direct transfer of ions and small molecules between adjacent cells. Gap junction channels are formed by integral membrane proteins called connexins, of which the best-studied isoform is connexin 43 (Cx43). Here we show that Cx43 is posttranslationally modified by SUMOylation. The data suggest that the SUMO system regulates the Cx43 protein level and the level of functional Cx43 gap junctions at the plasma membrane. Cx43 was found to be modified by SUMO-1, -2, and -3. Evidence is provided that the membrane-proximal lysines at positions 144 and 237, located in the Cx43 intracellular loop and C-terminal tail, respectively, act as SUMO conjugation sites. Mutations of lysine 144 or lysine 237 resulted in reduced Cx43 SUMOylation and reduced Cx43 protein and gap junction levels. Altogether, these data identify Cx43 as a SUMOylation target protein and represent the first evidence that gap junctions are regulated by the SUMO system.

Association of CD30 transcripts with Th1 responses and proinflammatory cytokines in patients with end-stage renal disease.

PubMed

Velásquez, Sonia Y; Opelz, Gerhard; Rojas, Mauricio; Süsal, Caner; Alvarez, Cristiam M

2016-05-01

High serum sCD30 levels are associated with inflammatory disorders and poor outcome in renal transplantation. The contribution to these phenomena of transcripts and proteins related to CD30-activation and -cleavage is unknown. We assessed in peripheral blood of end-stage renal disease patients (ESRDP) transcripts of CD30-activation proteins CD30 and CD30L, CD30-cleavage proteins ADAM10 and ADAM17, and Th1- and Th2-type immunity-related factors t-bet and GATA3. Additionally, we evaluated the same transcripts and release of sCD30 and 32 cytokines after allogeneic and polyclonal T-cell activation. In peripheral blood, ESRDP showed increased levels of t-bet and GATA3 transcripts compared to healthy controls (HC) (both P<0.01) whereas levels of CD30, CD30L, ADAM10 and ADAM17 transcripts were similar. Polyclonal and allogeneic stimulation induced higher levels of CD30 transcripts in ESRDP than in HC (both P<0.001). Principal component analysis (PCA) in allogeneic cultures of ESRDP identified two correlation clusters, one consisting of sCD30, the Th-1 cytokine IFN-γ, MIP-1α, RANTES, sIL-2Rα, MIP-1β, TNF-β, MDC, GM-CSF and IL-5, and another one consisting of CD30 and t-bet transcripts, IL-13 and proinflammatory proteins IP-10, IL-8, IL-1Rα and MCP-1. Reflecting an activated immune state, ESRDP exhibited after allostimulation upregulation of CD30 transcripts in T cells, which was associated with Th1 and proinflammatory responses. Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation.

PubMed

Desantis, Agata; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta; Fanciulli, Maurizio; Passananti, Claudio; Corbi, Nicoletta

2009-02-01

Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts.

PubMed

Jaquemar, D; Schenker, T; Trueb, B

1999-03-12

We have identified a novel transformation-sensitive mRNA, which is present in cultured fibroblasts but is lacking in SV40 transformed cells as well as in many mesenchymal tumor cell lines. The corresponding gene is located on human chromosome 8 in band 8q13. The open reading frame of the mRNA encodes a protein of 1119 amino acids forming two distinct domains. The N-terminal domain consists of 18 repeats that are related to the cytoskeletal protein ankyrin. The C-terminal domain contains six putative transmembrane segments that resemble many ion channels. This overall structure is reminiscent of TRP-like proteins that function as store-operated calcium channels. The novel protein with an Mr of 130 kDa is expressed at a very low level in human fibroblasts and at a moderate level in liposarcoma cells. Overexpression in eukaryotic cells appears to interfere with normal growth, suggesting that it might play a direct or indirect role in signal transduction and growth control.

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. © 2013.

Prospects for pro-environmental protein consumption in Europe: Cultural, culinary, economic and psychological factors.

PubMed

de Boer, Joop; Aiking, Harry

2018-02-01

The current ratio between plant and animal protein in the Western diet is causing serious threats to both public health and the environment. Healthy, pro-environmental protein consumption requires a transition to a diet with more plant protein and considerably less animal protein. The present paper focuses on the prospects of this transition by analyzing consumer responses to some key options in the context of regional differences across Europe. The aim is to assess how responses to the options might be shaped by 1) cultural, culinary and economic spatial gradients (including GDP per capita) at regional level and 2) differences in environmental friendly behavior and gender at individual level. The study, covering all EU members in 2012, compares regional level statistics (food supply data) with individual level statistics (consumer survey data) and vice-versa. The south-north latitude gradient showed a decreasing trend in vegetable and pulse protein supplies and, in parallel, a decreasing trend in positive consumer responses to the key options, probably due to differences in meal experiences. The west-east longitude gradient showed decreasing levels of animal protein supplies and GDP per capita. Individuals' willingness to do something positive for the environment and their gender played a weak but consistent role in the responses. To effectively stimulate diet changes, it is important to seek ways in which culinary and environmental aspects can complement each other and to ensure that diet changes do not depend solely on individual decisions but become an integral part of regional social processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Predicting protein-protein interactions on a proteome scale by matching evolutionary and structural similarities at interfaces using PRISM.

PubMed

Tuncbag, Nurcan; Gursoy, Attila; Nussinov, Ruth; Keskin, Ozlem

2011-08-11

Prediction of protein-protein interactions at the structural level on the proteome scale is important because it allows prediction of protein function, helps drug discovery and takes steps toward genome-wide structural systems biology. We provide a protocol (termed PRISM, protein interactions by structural matching) for large-scale prediction of protein-protein interactions and assembly of protein complex structures. The method consists of two components: rigid-body structural comparisons of target proteins to known template protein-protein interfaces and flexible refinement using a docking energy function. The PRISM rationale follows our observation that globally different protein structures can interact via similar architectural motifs. PRISM predicts binding residues by using structural similarity and evolutionary conservation of putative binding residue 'hot spots'. Ultimately, PRISM could help to construct cellular pathways and functional, proteome-scale annotation. PRISM is implemented in Python and runs in a UNIX environment. The program accepts Protein Data Bank-formatted protein structures and is available at http://prism.ccbb.ku.edu.tr/prism_protocol/.

mapDIA: Preprocessing and statistical analysis of quantitative proteomics data from data independent acquisition mass spectrometry.

PubMed

Teo, Guoshou; Kim, Sinae; Tsou, Chih-Chiang; Collins, Ben; Gingras, Anne-Claude; Nesvizhskii, Alexey I; Choi, Hyungwon

2015-11-03

Data independent acquisition (DIA) mass spectrometry is an emerging technique that offers more complete detection and quantification of peptides and proteins across multiple samples. DIA allows fragment-level quantification, which can be considered as repeated measurements of the abundance of the corresponding peptides and proteins in the downstream statistical analysis. However, few statistical approaches are available for aggregating these complex fragment-level data into peptide- or protein-level statistical summaries. In this work, we describe a software package, mapDIA, for statistical analysis of differential protein expression using DIA fragment-level intensities. The workflow consists of three major steps: intensity normalization, peptide/fragment selection, and statistical analysis. First, mapDIA offers normalization of fragment-level intensities by total intensity sums as well as a novel alternative normalization by local intensity sums in retention time space. Second, mapDIA removes outlier observations and selects peptides/fragments that preserve the major quantitative patterns across all samples for each protein. Last, using the selected fragments and peptides, mapDIA performs model-based statistical significance analysis of protein-level differential expression between specified groups of samples. Using a comprehensive set of simulation datasets, we show that mapDIA detects differentially expressed proteins with accurate control of the false discovery rates. We also describe the analysis procedure in detail using two recently published DIA datasets generated for 14-3-3β dynamic interaction network and prostate cancer glycoproteome. The software was written in C++ language and the source code is available for free through SourceForge website http://sourceforge.net/projects/mapdia/.This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

PubMed

He, Ding; Pengtao, Gong; Ju, Yang; Jianhua, Li; He, Li; Guocai, Zhang; Xichen, Zhang

2017-04-01

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis , we detected 2 strains of T. vaginalis ; the virus-infected (V + ) and uninfected (V - ) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V + compared with V - isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V + isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V + and V - isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, T.; Niepel, M.; McDermott, J. E.

It is not known whether cancer cells generally show quantitative differences in the expression of signaling pathway proteins that could dysregulate signal transduction. To explore this issue, we first defined the primary components of the EGF-MAPK pathway in normal human mammary epithelial cells, identifying 16 core proteins and 10 feedback regulators. We then quantified their absolute abundance across a panel of normal and cancer cell lines. We found that core pathway proteins were expressed at very similar levels across all cell types. In contrast, the EGFR and transcriptionally controlled feedback regulators were expressed at highly variable levels. The absolute abundancemore » of most core pathway proteins was between 50,000- 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower levels (2,000-5,000 per cell). MAPK signaling showed saturation in all cells between 3,000-10,000 occupied EGFR, consistent with the idea that low adaptor levels limit signaling. Our results suggest that the core MAPK pathway is essentially invariant across different cell types, with cell- specific differences in signaling likely due to variable levels of feedback regulators. The low abundance of adaptors relative to the EGFR could be responsible for previous observation of saturable signaling, endocytosis, and high affinity EGFR.« less

Effects of a high plant protein diet on the somatotropic system and cholecystokinin in Atlantic salmon (Salmo salar L.).

PubMed

Hevrøy, Ernst M; El-Mowafi, Adel; Taylor, Richard; Norberg, Birgitta; Espe, Marit

2008-12-01

To investigate the endocrine signalling from dietary plant protein on somatotropic system and gastrointestinal hormone cholecystokinin (CCK), two iso-amino acid diets based on either high plant or high fish meal protein were fed to Atlantic salmon. Salmon with an average starting weight of 641+/-23 g (N=180), were fed a fish meal (FM) based diet (containing 40% FM) or diets mainly consisting of blended plant proteins (PP) containing only 13% marine protein, of which only 5% was FM for 3 months. mRNA levels of target genes GH, GH-R, IGF-I, IGF-II, IGFBP-1, IGF-IR in addition to CCK-L, were studied in brain, hepatic tissue and fast muscle, and circulating levels of IGF-I in plasma of Atlantic salmon were measured. We detected reduced feed intake resulting in lower growth, weight gain and muscle protein accretion in salmon fed plant protein compared to a diet based on fish meal. There were no significant effects on the regulation of the target genes in brain or in hepatic tissues, but a trend of down-regulation of IGF-I was detected in fast muscle. Lower feed intake, and therefore lower intake of the indispensable amino acids, may have resulted in lower pituitary GH and lower IGF-I mRNA levels in muscle tissues. This, together with higher protein catabolism, may be the main cause of the reduced growth of salmon fed plant protein diet. There were no signalling effects detected either by the minor differences of the diets on mRNA levels of GH, GH-R, IGF-IR, IGF-II, IGFBP-1, CCK or plasma protein IGF-I.

Opposite Dysregulation of Fragile-X Mental Retardation Protein and Heteronuclear Ribonucleoprotein C Protein Associates with Enhanced APP Translation in Alzheimer Disease.

PubMed

Borreca, Antonella; Gironi, Katia; Amadoro, Giusy; Ammassari-Teule, Martine

2016-07-01

Amyloid precursor protein (APP) is overexpressed in familiar and sporadic Alzheimer Disease (AD) patients suggesting that, in addition to abnormalities in APP cleavage, enhanced levels of APP full length might contribute to the pathology. Based on data showing that the two RNA binding proteins (RBPs), Fragile-X Mental Retardation Protein (FMRP) and heteronuclear Ribonucleoprotein C (hnRNP C), exert an opposite control on APP translation, we have analyzed whether expression and translation of these two RBPs vary in relation to changes in APP protein and mRNA levels in the AD brain at 1, 3, and 6 months of age. Here, we show that, as expected, human APP is overexpressed in hippocampal total extract from Tg2576 mice at all age points. APP overexpression, however, is not stable over time but reaches its maximal level in 1-month-old mutants in association with the stronger (i) reduction of FMRP and (ii) augmentation of hnRNP C. APP levels then decrease progressively as a function of age in close relationship with the gradual normalization of FMRP and hnRNP C levels. Consistent with the mouse data, expression of FMRP and hnRNP C are, respectively, decreased and increased in hippocampal synaptosomes from sporadic AD patients. Our findings identify two RBP targets that might be manipulated for reducing abnormally elevated levels of APP in the AD brain, with the hypothesis that acting upstream of amyloidogenic processing might contribute to attenuate the amyloid burden.

[Level of selected antibacterial tear proteins in children with diabetes type 1].

PubMed

Moll, Agnieszka; Wyka, Krystyna; Młynarski, Wojciech; Niwald, Anna

2011-01-01

Antibacterial immunity in diabetes is impaired, which increases the risk of general and local infections. The aim of the study was to evaluate non-specific local antibacterial immunity based on lactoferrin and lysozyme concentration in tears in children with diabetes type 1. Children at the age of 10-18 years old were studied. Group 1. consisted of children without diabetes, group 2. included patients with new onset of diabetes and group 3. consisted of children with decade-long diabetes. Among all patients tears were collected from inferior coniunctival fornix with hematocrit glass capillaries in purpose to measure lactoferrin and lysozyme concentration. ELISA method was used in laboratory testing. Level of lactoferrin did not differ significantly among all groups. Concentration of lysozyme was statistically lower in group with decade-long diabetes (group 3.) compared to patients without diabetes. Mild correlation between lactoferrin and lysozyme levels was seen in individual patients in whole group of probands together. Diabetes type 1 in children is associated with significant changes in concentration of tear proteins, which contribute to antibacterial immunity.

Comparison of fumonisin contamination using HPLC and ELISA methods in bt and near-isogenic maize hybrids infested with European corn borer or western bean cutworm.

PubMed

Bowers, Erin; Hellmich, Richard; Munkvold, Gary

2014-07-09

Field trials were conducted from 2007 to 2010 to compare grain fumonisin levels among non-Bt maize hybrids and Bt hybrids with transgenic protection against manual infestations of European corn borer (ECB) and Western bean cutworm (WBC). HPLC and ELISA were used to measure fumonisin levels. Results of the methods were highly correlated, but ELISA estimates were higher. Bt hybrids experienced less insect injury, Fusarium ear rot, and fumonisin contamination compared to non-Bt hybrids. WBC infestation increased fumonisin content compared to natural infestation in non-Bt and hybrids expressing Cry1Ab protein in five of eight possible comparisons; in Cry1F hybrids, WBC did not impact fumonisins. These results indicate that WBC is capable of increasing fumonisin levels in maize. Under WBC infestation, Cry1F mitigated this risk more consistently than Cry1Ab or non-Bt hybrids. Transgenically expressed Bt proteins active against multiple lepidopteran pests can provide broad, consistent reductions in the risk of fumonisin contamination.

Purification and characterization of a human pancreatic adenocarcinoma mucin.

PubMed

Khorrami, Ali M; Choudhury, Amit; Andrianifahanana, Mahefatiana; Varshney, Grish C; Bhattacharyya, Sambhu N; Hollingsworth, Michael A; Kaufman, Bernard; Batra, Surinder K

2002-01-01

Pancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.

Changes of Protein Turnover in Aging Caenorhabditis elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dhondt, Ineke; Petyuk, Vladislav A.; Bauer, Sophie

Protein turnover rates severely decline in aging organisms, including C. elegans. However, limited information is available on turnover dynamics at the individual protein level during aging. We followed changes in protein turnover at one-day resolution using a multiple-pulse 15Nlabeling and accurate mass spectrometry approach. Forty percent of the proteome shows gradual slowdown in turnover with age, whereas only few proteins show increased turnover. Decrease in protein turnover was consistent for only a minority of functionally related protein subsets, including tubulins and vitellogenins, whereas randomly diverging turnover patterns with age were the norm. Our data suggests increased heterogeneity of protein turnovermore » of the translation machinery, whereas protein turnover of ubiquitin-proteasome and antioxidant systems are well-preserved over time. Hence, we presume that maintenance of quality control mechanisms is a protective strategy in aging worms, although the ultimate proteome collapse is inescapable.« less

Arginine supplementation modulates pig plasma lipids, but not hepatic fatty acids, depending on dietary protein level with or without leucine.

PubMed

Madeira, Marta Sofia Morgado Dos Santos; Rolo, Eva Sofia Alves; Pires, Virgínia Maria Rico; Alfaia, Cristina Maria Riscado Pereira Mateus; Coelho, Diogo Francisco Maurício; Lopes, Paula Alexandra Antunes Brás; Martins, Susana Isabel Vargas; Pinto, Rui Manuel Amaro; Prates, José António Mestre

2017-05-30

In the present study, the effect of arginine and leucine supplementation, and dietary protein level, were investigated in commercial crossbred pigs to clarify their individual or combined impact on plasma metabolites, hepatic fatty acid composition and mRNA levels of lipid sensitive factors. The experiment was conducted on fifty-four entire male pigs (Duroc × Pietrain × Large White × Landrace crossbred) from 59 to 92 kg of live weight. Each pig was randomly assigned to one of six experimental treatments (n = 9). The treatments followed a 2 × 3 factorial arrangement, providing two levels of arginine supplementation (0 vs. 1%) and three levels of basal diet (normal protein diet, NPD; reduced protein diet, RPD; reduced protein diet with 2% of leucine, RPDL). Significant interactions between arginine supplementation and protein level were observed across plasma lipids. While dietary arginine increased total lipids, total cholesterol, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol and triacylglycerols in NPD, the inverse effect was observed in RPD. Overall, dietary treatments had a minor impact on hepatic fatty acid composition. RPD increased 18:1c9 fatty acid while the combination of leucine and RPD reduced 18:0 fatty acid. Arginine supplementation increased the gene expression of FABP1, which contributes for triacylglycerols synthesis without affecting hepatic fatty acids content. RPD, with or without leucine addition, upregulated the lipogenic gene CEBPA but downregulated the fat oxidation gene LPIN1. Arginine supplementation was responsible for a modulated effect on plasma lipids, which is dependent on dietary protein level. It consistently increased lipaemia in NPD, while reducing the correspondent metabolites in RPD. In contrast, arginine had no major impact, neither on hepatic fatty acids content nor on fatty acid composition. Likewise, leucine supplementation of RPD, regardless the presence of arginine, promoted no changes on total fatty acids in the liver. Ultimately, arginine, leucine and dietary protein reduction seem to be unrelated with fatty liver development.

Decreased function of survival motor neuron protein impairs endocytic pathways.

PubMed

Dimitriadi, Maria; Derdowski, Aaron; Kalloo, Geetika; Maginnis, Melissa S; O'Hern, Patrick; Bliska, Bryn; Sorkaç, Altar; Nguyen, Ken C Q; Cook, Steven J; Poulogiannis, George; Atwood, Walter J; Hall, David H; Hart, Anne C

2016-07-26

Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death.

Decreased function of survival motor neuron protein impairs endocytic pathways

PubMed Central

Dimitriadi, Maria; Derdowski, Aaron; Kalloo, Geetika; Maginnis, Melissa S.; O’Hern, Patrick; Bliska, Bryn; Sorkaç, Altar; Nguyen, Ken C. Q.; Cook, Steven J.; Poulogiannis, George; Atwood, Walter J.; Hall, David H.; Hart, Anne C.

2016-01-01

Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death. PMID:27402754

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

PubMed

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

2018-02-01

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

A two-site ELISA can quantify upregulation of SMN protein by drugs for spinal muscular atrophy.

PubMed

Nguyen thi Man; Humphrey, E; Lam, L T; Fuller, H R; Lynch, T A; Sewry, C A; Goodwin, P R; Mackenzie, A E; Morris, G E

2008-11-25

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons during early or postnatal development. Severity is variable and is inversely related to the levels of survival of motor neurons (SMN) protein. The aim of this study was to produce a two-site ELISA capable of measuring both the low, basal levels of SMN protein in cell cultures from patients with severe SMA and small increases in these levels after treatment of cells with drugs. A monoclonal antibody against recombinant SMN, MANSMA1, was selected for capture of SMN onto microtiter plates. A selected rabbit antiserum against refolded recombinant SMN was used for detection of the captured SMN. The ratio of SMN levels in control fibroblasts to levels in SMA fibroblasts was greater than 3.0, consistent with Western blot data. The limit of detection was 0.13 ng/mL and SMN could be measured in human NT-2 neuronal precursor cells grown in 96-well culture plates (3 x 10(4) cells per well). Increases in SMN levels of 50% were demonstrable by ELISA after 24 hours treatment of 10(5) SMA fibroblasts with valproate or phenylbutyrate. A rapid and specific two-site, 96-well ELISA assay, available in kit format, can now quantify the effects of drugs on survival of motor neurons protein levels in cell cultures.

In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes

PubMed Central

1986-01-01

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick- translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling. PMID:3084498

Muscle damage induced by stretch-shortening cycle exercise.

PubMed

Kyröläinen, H; Takala, T E; Komi, P V

1998-03-01

Strenuous stretch-shortening cycle exercise was used as a model to study the leakage of proteins from skeletal muscle. The analysis included serum levels of creatine kinase (S-CK), myoglobin (S-Mb), and carbonic anhydrase (S-CA III). Blood samples from power- (N=11) and endurance-trained (N=10) athletes were collected before, 0, and 2 h after the exercise, which consisted of a total of 400 jumps. The levels of all determined myocellular proteins increased immediately after the exercise (P < 0.05-0.001) among both subject groups. In the endurance group, the protein levels increased (P < 0.05-0.001) further during the following 2 h after the exercise, and the ratio of S-CA III and S-Mb decreased (P < 0.05) in a before-after comparison. This was not the case among the power group despite their greater mechanical work (P < 0.001) and higher ratio of eccentric and concentric EMG activity of the leg extensor muscles (P < 0.05). The differences of the determined protein levels between the subject groups might be due to obvious differences in the muscle fiber distribution, differences in recruitment order of motor units, and/or differences in training background.

Presence of undeclared peanut protein in chocolate bars imported from Europe.

PubMed

Vadas, Peter; Perelman, Boris

2003-10-01

Peanut allergens are both stable and potent and are capable of inducing anaphylactic reactions at low concentrations. Consequently, the consumption of peanuts remains the most common cause of food-induced anaphylactic death. Since accidental exposure to peanuts is a common cause of potentially fatal anaphylaxis in peanut-allergic individuals, we tested for the presence of peanut protein in chocolate bars produced in Europe and North America that did not list peanuts as an ingredient. Ninety-two chocolate bars, of which 32 were manufactured in North America and 60 were imported from Europe, were tested by the Veratox assay. None of the 32 North American chocolate products, including 19 with precautionary labeling, contained detectable peanut protein. In contrast, 30.8% of products from western Europe without precautionary labeling contained detectable levels of peanut protein. Sixty-two percent of products from eastern Europe without precautionary labeling contained detectable peanut protein at levels of up to 245 ppm. The absence of precautionary labeling and the absence of the declaration of "peanut" as an ingredient in chocolate bars made in eastern and central Europe were not found to guarantee that these products were actually free of contaminating peanut protein. In contrast, North American manufacturers have attained a consistent level of safety and reliability for peanut-allergic consumers.

Repeated measurement of nasal lavage fluid chemokines in school-age children with asthma.

PubMed

Noah, Terry L; Tudor, Gail E; Ivins, Sally S; Murphy, Paula C; Peden, David B; Henderson, Frederick W

2006-02-01

Inflammatory processes at the mucosal surface may play a role in maintenance of asthma pathophysiology. Cross-sectional studies in asthmatic patients suggest that chemokines such as interleukin 8 (IL-8) are overproduced by respiratory epithelium. To test the hypothesis that chemokine levels are persistently elevated in the respiratory secretions of asthmatic children at a stable baseline. We measured nasal lavage fluid (NLF) levels of chemokines and other mediators at 3- to 4-month intervals in a longitudinal study of asthmatic children, with nonasthmatic siblings as controls. In a linear mixed-model analysis, both family and day of visit had significant effects on nasal mediators. Thus, data for 12 asthmatic-nonasthmatic sibling pairs who had 3 or more same-day visits were analyzed separately. For sibling pairs, median eosinophil cationic protein levels derived from serial measurements in NLF were elevated in asthmatic patients compared with nonasthmatic patients, with a near-significant tendency for elevation of total protein and eotaxin levels as well. However, no significant differences were found for IL-8 or several other chemokines. Ratios of IL-13 or IL-5 to interferon-gamma released by house dust mite antigen-stimulated peripheral blood mononuclear cells, tested on a single occasion, were significantly increased for asthmatic patients. Substantial temporal and family-related variability exists in nasal inflammation in asthmatic children. Although higher levels of eosinophil cationic protein are usually present in NLF of patients with stable asthma compared with patients without asthma, chemokines other than eotaxin are not consistently increased. Eosinophil activation at the mucosal surface is a more consistent predictor of asthmatic symptoms than nonspecific elevation of epithelium-derived inflammatory chemokine levels.

Pollen feeding proteomics: Salivary proteins of the passion flower butterfly, Heliconius melpomene.

PubMed

Harpel, Desiree; Cullen, Darron A; Ott, Swidbert R; Jiggins, Chris D; Walters, James R

2015-08-01

While most adult Lepidoptera use flower nectar as their primary food source, butterflies in the genus Heliconius have evolved the novel ability to acquire amino acids from consuming pollen. Heliconius butterflies collect pollen on their proboscis, moisten the pollen with saliva, and use a combination of mechanical disruption and chemical degradation to release free amino acids that are subsequently re-ingested in the saliva. Little is known about the molecular mechanisms of this complex pollen feeding adaptation. Here we report an initial shotgun proteomic analysis of saliva from Heliconius melpomene. Results from liquid-chromatography tandem mass-spectrometry confidently identified 31 salivary proteins, most of which contained predicted signal peptides, consistent with extracellular secretion. Further bioinformatic annotation of these salivary proteins indicated the presence of four distinct functional classes: proteolysis (10 proteins), carbohydrate hydrolysis (5), immunity (6), and "housekeeping" (4). Additionally, six proteins could not be functionally annotated beyond containing a predicted signal sequence. The presence of several salivary proteases is consistent with previous demonstrations that Heliconius saliva has proteolytic capacity. It is likely that these proteins play a key role in generating free amino acids during pollen digestion. The identification of proteins functioning in carbohydrate hydrolysis is consistent with Heliconius butterflies consuming nectar, like other lepidopterans, as well as pollen. Immune-related proteins in saliva are also expected, given that ingestion of pathogens is a likely route to infection. The few "housekeeping" proteins are likely not true salivary proteins and reflect a modest level of contamination that occurred during saliva collection. Among the unannotated proteins were two sets of paralogs, each seemingly the result of a relatively recent tandem duplication. These results offer a first glimpse into the molecular foundation of Heliconius pollen feeding and provide a substantial advance towards comprehensively understanding this striking evolutionary novelty. Copyright © 2015 Elsevier Ltd. All rights reserved.

Newborn Mouse Lens Proteome and Its Alteration by Lysine 6 Mutant Ubiquitin

PubMed Central

2015-01-01

Ubiquitin is a tag that often initiates degradation of proteins by the proteasome in the ubiquitin proteasome system. Targeted expression of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in lens development and cataract formation, suggesting critical functions for ubiquitin in lens. To study the developmental processes that require intact ubiquitin, we executed the most extensive characterization of the lens proteome to date. We quantified lens protein expression changes in multiple replicate pools of P1 wild-type and K6W-Ub-expressing mouse lenses. Lens proteins were digested with trypsin, peptides were separated using strong cation exchange and reversed-phase liquid chromatography, and tandem mass (MS/MS) spectra were collected with a linear ion trap. Transgenic mice that expressed low levels of K6W-Ub (low expressers) had normal, clear lenses at birth, whereas the lenses that expressed high levels of K6W-Ub (higher expressers) had abnormal lenses and cataracts at birth. A total of 2052 proteins were identified, of which 996 were reliably quantified and compared between wild-type and K6W-Ub transgenic mice. Consistent with a delayed developmental program, fiber-cell-specific proteins, such as γ-crystallins (γA, γB, γC, and γE), were down-regulated in K6W-Ub higher expressers. Up-regulated proteins were involved in energy metabolism, signal transduction, and proteolysis. The K6W-Ub low expressers exhibited delayed onset and milder cataract consistent with smaller changes in protein expression. Because lens protein expression changes occurred prior to lens morphological abnormalities and cataract formation in K6W-Ub low expressers, it appears that expression of K6W-Ub sets in motion a process of altered protein expression that results in developmental defects and cataract. PMID:24450463

XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons.

PubMed

Wang, Hung-Li; Chang, Ning-Chun; Weng, Yi-Hsin; Yeh, Tu-Hsueh

2013-04-01

Truncating or missense mutation of cullin 4B (CUL4B) is one of the most prevalent causes underlying X-linked intellectual disability (XLID). CUL4B-RING E3 ubiquitin ligase promotes ubiquitination and degradation of various proteins. Consistent with previous studies, overexpression of wild-type CUL4B in 293 cells enhanced ubiquitylation and degradation of TSC2 or cyclin E. The present study shows that XLID mutant (R388X), (R572C) or (V745A) CULB failed to promote ubiquitination and degradation of TSC2 or cyclin E. Adenoviruses-mediated expression of wild-type CUL4B decreased protein level of TSC2 or cyclin E in cultured neocortical neurons of frontal lobe. Furthermore, shRNA-mediated CUL4B knockdown caused an upregulation of TSC2 or cyclin E. XLID mutant (R388X), (R572C) or (V745A) CUL4B did not downregulate protein expression of TSC2 or cyclin E in neocortical neurons. By promoting TSC2 degradation, CUL4B could positively regulate mTOR activity in neocortical neurons of frontal cortex. Consistent with this hypothesis, CUL4B knockdown-induced upregulation of TSC2 in neocortical neurons resulted in a decreased protein level of active phospho-mTOR(Ser2448) and a reduced expression of active phospho-p70S6K(Thr389) and phospho-4E-BP1(Thr37/46), two main substrates of mTOR-mediated phosphorylation. Wild-type CUL4B also increased protein level of active phospho-mTOR(Ser2448), phospho-p70S6K(Thr389) or phospho-4E-BP1(Thr37/46). XLID CUL4B mutants did not affect protein level of active phospho-mTOR(Ser2448), phospho-p70S6K(Thr389) or phospho-4E-BP1(Thr37/46). Our results suggest that XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons. Copyright © 2013 Elsevier B.V. All rights reserved.

Functional Tat transport of unstructured, small, hydrophilic proteins.

PubMed

Richter, Silke; Lindenstrauss, Ute; Lücke, Christian; Bayliss, Richard; Brüser, Thomas

2007-11-16

The twin-arginine translocation (Tat) system is a protein translocation system that is adapted to the translocation of folded proteins across biological membranes. An understanding of the folding requirements for Tat substrates is of fundamental importance for the elucidation of the transport mechanism. We now demonstrate for the first time Tat transport for fully unstructured proteins, using signal sequence fusions to naturally unfolded FG repeats from the yeast Nsp1p nuclear pore protein. The transport of unfolded proteins becomes less efficient with increasing size, consistent with only a single interaction between the system and the substrate. Strikingly, the introduction of six residues from the hydrophobic core of a globular protein completely blocked translocation. Physiological data suggest that hydrophobic surface patches abort transport at a late stage, most likely by membrane interactions during transport. This study thus explains the observed restriction of the Tat system to folded globular proteins on a molecular level.

Reproducibility and consistency of proteomic experiments on natural populations of a non-model aquatic insect.

PubMed

Hidalgo-Galiana, Amparo; Monge, Marta; Biron, David G; Canals, Francesc; Ribera, Ignacio; Cieslak, Alexandra

2014-01-01

Population proteomics has a great potential to address evolutionary and ecological questions, but its use in wild populations of non-model organisms is hampered by uncontrolled sources of variation. Here we compare the response to temperature extremes of two geographically distant populations of a diving beetle species (Agabus ramblae) using 2-D DIGE. After one week of acclimation in the laboratory under standard conditions, a third of the specimens of each population were placed at either 4 or 27°C for 12 h, with another third left as a control. We then compared the protein expression level of three replicated samples of 2-3 specimens for each treatment. Within each population, variation between replicated samples of the same treatment was always lower than variation between treatments, except for some control samples that retained a wider range of expression levels. The two populations had a similar response, without significant differences in the number of protein spots over- or under-expressed in the pairwise comparisons between treatments. We identified exemplary proteins among those differently expressed between treatments, which proved to be proteins known to be related to thermal response or stress. Overall, our results indicate that specimens collected in the wild are suitable for proteomic analyses, as the additional sources of variation were not enough to mask the consistency and reproducibility of the response to the temperature treatments.

ATP5B and ETFB metabolic markers in children with congenital hydronephrosis.

PubMed

Zhao, Qi; Yang, Yi; Wang, Changlin; Hou, Ying; Chen, Hui

2016-12-01

Congenital obstructive nephropathy is the primary cause of chronic renal failure in children. Disorders of mitochondrial energy metabolism may be a primary factor underlying tubular cell apoptosis in hydronephrosis. The β-F1-ATPase (ATP5B) and electron transfer flavoprotein β subunit (ETFB) metabolic markers are involved in mitochondrial energy metabolism in other diseases. The aim of the present study was to evaluate whether ATP5B and ETFB are represented in the hydronephrotic kidney, and whether they are associated with the progression of hydronephrosis. The cohort examined consisted of 20 children with hydronephrosis, graded III and IV using the Society for Fetal Urology grading system, and a control group consisting of 20 patients with nephroblastoma. Reverse transcription‑quantitative polymerase chain reaction and immunoblot analyses were used to investigate the differential expression of genes and proteins in the two groups. The gene and protein expression levels of ATP5B and ETFB were upregulated in the hydronephrosis group. Correlation analyses revealed negative correlations between ATP5B, ETFB protein and split renal function (SRF). Receiver‑operator curve analysis found a diagnostic profile of the ETFB protein in identifying children with hydronephrosis with abnormal SRF (<45%). These results suggested that increasing levels of ATP5B and ETFB were associated with worsening renal injury. ATP5B and ETFB may be novel markers in hydronephrosis and require further detailed investigation.

ATP5B and ETFB metabolic markers in children with congenital hydronephrosis

PubMed Central

Zhao, Qi; Yang, Yi; Wang, Changlin; Hou, Ying; Chen, Hui

2016-01-01

Congenital obstructive nephropathy is the primary cause of chronic renal failure in children. Disorders of mitochondrial energy metabolism may be a primary factor underlying tubular cell apoptosis in hydronephrosis. The β-F1-ATPase (ATP5B) and electron transfer flavoprotein β subunit (ETFB) metabolic markers are involved in mitochondrial energy metabolism in other diseases. The aim of the present study was to evaluate whether ATP5B and ETFB are represented in the hydronephrotic kidney, and whether they are associated with the progression of hydronephrosis. The cohort examined consisted of 20 children with hydronephrosis, graded III and IV using the Society for Fetal Urology grading system, and a control group consisting of 20 patients with nephroblastoma. Reverse transcription-quantitative polymerase chain reaction and immunoblot analyses were used to investigate the differential expression of genes and proteins in the two groups. The gene and protein expression levels of ATP5B and ETFB were upregulated in the hydronephrosis group. Correlation analyses revealed negative correlations between ATP5B, ETFB protein and split renal function (SRF). Receiver-operator curve analysis found a diagnostic profile of the ETFB protein in identifying children with hydronephrosis with abnormal SRF (<45%). These results suggested that increasing levels of ATP5B and ETFB were associated with worsening renal injury. ATP5B and ETFB may be novel markers in hydronephrosis and require further detailed investigation. PMID:27840937

Mixed Tocotrienols Inhibit Prostate Carcinogenesis in TRAMP Mice

PubMed Central

Barve, Avantika; Khor, Tin Oo; Reuhl, Kenneth; Reddy, Bandaru; Newmark, Harold; Kong, Ah-Ng

2015-01-01

The biological activities of tocotrienols are receiving increasing attention. Herein, we report the efficacy of a mixed-tocotrienol diet against prostate tumorigenesis in the transgenic adenocarcinoma mouse prostate (TRAMP) mouse model. Male TRAMP mice, 8 wk old, were fed 0.1%, 0.3%, or 1% mixed tocotrienols in AIN-76A diet up to 24 wk old. Likewise, a positive control group consisting of male TRAMP mice and a negative control group consisting of wild-type nontransgenic mice were fed regular AIN-76A diet up to 24 wk old. Our results show that mixed-tocotrienol-fed groups had a lower incidence of tumor formation along with a significant reduction in the average wet weight of genitourinary apparatus. Furthermore, mixed tocotrienols significantly reduced the levels of high-grade neoplastic lesions as compared to the positive controls. This decrease in levels of high-grade neoplastic lesions was found to be associated with increased expression of proapoptotic proteins BAD (Bcl2 antagonist of cell death) and cleaved caspase-3 and cell cycle regulatory proteins cyclin dependent kinase inhibitors p21 and p27. In contrast, the expression of cyclins A and E were found to be decreased in mixed-tocotrienol groups. Taken together, our results show that by modulating cell cycle regulatory proteins and increasing expression of proapoptotic proteins, mixed tocotrienols suppress prostate tumorigenesis in the TRAMP mice. PMID:20661828

Reproducibility and Consistency of Proteomic Experiments on Natural Populations of a Non-Model Aquatic Insect

PubMed Central

Hidalgo-Galiana, Amparo; Monge, Marta; Biron, David G.; Canals, Francesc; Ribera, Ignacio; Cieslak, Alexandra

2014-01-01

Population proteomics has a great potential to address evolutionary and ecological questions, but its use in wild populations of non-model organisms is hampered by uncontrolled sources of variation. Here we compare the response to temperature extremes of two geographically distant populations of a diving beetle species (Agabus ramblae) using 2-D DIGE. After one week of acclimation in the laboratory under standard conditions, a third of the specimens of each population were placed at either 4 or 27°C for 12 h, with another third left as a control. We then compared the protein expression level of three replicated samples of 2–3 specimens for each treatment. Within each population, variation between replicated samples of the same treatment was always lower than variation between treatments, except for some control samples that retained a wider range of expression levels. The two populations had a similar response, without significant differences in the number of protein spots over- or under-expressed in the pairwise comparisons between treatments. We identified exemplary proteins among those differently expressed between treatments, which proved to be proteins known to be related to thermal response or stress. Overall, our results indicate that specimens collected in the wild are suitable for proteomic analyses, as the additional sources of variation were not enough to mask the consistency and reproducibility of the response to the temperature treatments. PMID:25133588

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

PubMed

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

2015-01-15

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

Effect of dietary α-lipoic acid on the mRNA expression of genes involved in drug metabolism and antioxidation system in rat liver.

PubMed

Ide, Takashi

2014-08-14

In the present study, the mRNA levels of hepatic proteins involved in the drug metabolism of rats fed α-lipoic acid were evaluated by DNA microarray and real-time PCR analyses. Experimental diets containing 0, 0·1, 0·25 and 0·5 % (w/w) α-lipoic acid were fed to four groups of rats consisting of seven animals each for 21 d. DNA microarray analysis revealed that the diet containing 0·5 % α-lipoic acid significantly (P< 0·05) increased the mRNA levels of various phase I drug-metabolising enzymes up to 15-fold and phase II enzymes up to 52-fold in an isoenzyme-specific manner. α-Lipoic acid also up-regulated the mRNA levels of some members of the ATP-binding cassette transporter superfamily, presumed to be involved in the exportation of xenobiotics, up to 6·6-fold. In addition, we observed that α-lipoic acid increased the mRNA levels of many proteins involved in antioxidation, such as members of the thiol redox system (up to 5·5-fold), metallothioneins (up to 12-fold) and haeme oxygenase 1 (1·5-fold). These results were confirmed using real-time PCR analysis, and α-lipoic acid dose dependently increased the mRNA levels of various proteins involved in drug metabolism and antioxidation. Consistent with these observations, α-lipoic acid dose dependently increased the hepatic concentration of glutathione and the activities of glutathione reductase and glutathione transferase measured using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates, but decreased the hepatic and serum concentrations of malondialdehyde. In conclusion, the present study unequivocally demonstrated that α-lipoic acid increases the mRNA expression of proteins involved in drug metabolism and antioxidation in the liver.

Prolonged Fasting Identifies Heat Shock Protein 10 as a Sirtuin 3 Substrate

PubMed Central

Lu, Zhongping; Chen, Yong; Aponte, Angel M.; Battaglia, Valentina; Gucek, Marjan; Sack, Michael N.

2015-01-01

Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function. PMID:25505263

The Protein-DNA Interface database

PubMed Central

2010-01-01

The Protein-DNA Interface database (PDIdb) is a repository containing relevant structural information of Protein-DNA complexes solved by X-ray crystallography and available at the Protein Data Bank. The database includes a simple functional classification of the protein-DNA complexes that consists of three hierarchical levels: Class, Type and Subtype. This classification has been defined and manually curated by humans based on the information gathered from several sources that include PDB, PubMed, CATH, SCOP and COPS. The current version of the database contains only structures with resolution of 2.5 Å or higher, accounting for a total of 922 entries. The major aim of this database is to contribute to the understanding of the main rules that underlie the molecular recognition process between DNA and proteins. To this end, the database is focused on each specific atomic interface rather than on the separated binding partners. Therefore, each entry in this database consists of a single and independent protein-DNA interface. We hope that PDIdb will be useful to many researchers working in fields such as the prediction of transcription factor binding sites in DNA, the study of specificity determinants that mediate enzyme recognition events, engineering and design of new DNA binding proteins with distinct binding specificity and affinity, among others. Finally, due to its friendly and easy-to-use web interface, we hope that PDIdb will also serve educational and teaching purposes. PMID:20482798

The Protein-DNA Interface database.

PubMed

Norambuena, Tomás; Melo, Francisco

2010-05-18

The Protein-DNA Interface database (PDIdb) is a repository containing relevant structural information of Protein-DNA complexes solved by X-ray crystallography and available at the Protein Data Bank. The database includes a simple functional classification of the protein-DNA complexes that consists of three hierarchical levels: Class, Type and Subtype. This classification has been defined and manually curated by humans based on the information gathered from several sources that include PDB, PubMed, CATH, SCOP and COPS. The current version of the database contains only structures with resolution of 2.5 A or higher, accounting for a total of 922 entries. The major aim of this database is to contribute to the understanding of the main rules that underlie the molecular recognition process between DNA and proteins. To this end, the database is focused on each specific atomic interface rather than on the separated binding partners. Therefore, each entry in this database consists of a single and independent protein-DNA interface.We hope that PDIdb will be useful to many researchers working in fields such as the prediction of transcription factor binding sites in DNA, the study of specificity determinants that mediate enzyme recognition events, engineering and design of new DNA binding proteins with distinct binding specificity and affinity, among others. Finally, due to its friendly and easy-to-use web interface, we hope that PDIdb will also serve educational and teaching purposes.

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon

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.more » 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.« less

Integrated Proteomic Pipeline Using Multiple Search Engines for a Proteogenomic Study with a Controlled Protein False Discovery Rate.

PubMed

Park, Gun Wook; Hwang, Heeyoun; Kim, Kwang Hoe; Lee, Ju Yeon; Lee, Hyun Kyoung; Park, Ji Yeong; Ji, Eun Sun; Park, Sung-Kyu Robin; Yates, John R; Kwon, Kyung-Hoon; Park, Young Mok; Lee, Hyoung-Joo; Paik, Young-Ki; Kim, Jin Young; Yoo, Jong Shin

2016-11-04

In the Chromosome-Centric Human Proteome Project (C-HPP), false-positive identification by peptide spectrum matches (PSMs) after database searches is a major issue for proteogenomic studies using liquid-chromatography and mass-spectrometry-based large proteomic profiling. Here we developed a simple strategy for protein identification, with a controlled false discovery rate (FDR) at the protein level, using an integrated proteomic pipeline (IPP) that consists of four engrailed steps as follows. First, using three different search engines, SEQUEST, MASCOT, and MS-GF+, individual proteomic searches were performed against the neXtProt database. Second, the search results from the PSMs were combined using statistical evaluation tools including DTASelect and Percolator. Third, the peptide search scores were converted into E-scores normalized using an in-house program. Last, ProteinInferencer was used to filter the proteins containing two or more peptides with a controlled FDR of 1.0% at the protein level. Finally, we compared the performance of the IPP to a conventional proteomic pipeline (CPP) for protein identification using a controlled FDR of <1% at the protein level. Using the IPP, a total of 5756 proteins (vs 4453 using the CPP) including 477 alternative splicing variants (vs 182 using the CPP) were identified from human hippocampal tissue. In addition, a total of 10 missing proteins (vs 7 using the CPP) were identified with two or more unique peptides, and their tryptic peptides were validated using MS/MS spectral pattern from a repository database or their corresponding synthetic peptides. This study shows that the IPP effectively improved the identification of proteins, including alternative splicing variants and missing proteins, in human hippocampal tissues for the C-HPP. All RAW files used in this study were deposited in ProteomeXchange (PXD000395).

Transcriptional response of honey bee larvae infected with the bacterial pathogen Paenibacillus larvae.

PubMed

Cornman, Robert Scott; Lopez, Dawn; Evans, Jay D

2013-01-01

American foulbrood disease of honey bees is caused by the bacterium Paenibacillus larvae. Infection occurs per os in larvae and systemic infection requires a breaching of the host peritrophic matrix and midgut epithelium. Genetic variation exists for both bacterial virulence and host resistance, and a general immunity is achieved by larvae as they age, the basis of which has not been identified. To quickly identify a pool of candidate genes responsive to P. larvae infection, we sequenced transcripts from larvae inoculated with P. larvae at 12 hours post-emergence and incubated for 72 hours, and compared expression levels to a control cohort. We identified 75 genes with significantly higher expression and six genes with significantly lower expression. In addition to several antimicrobial peptides, two genes encoding peritrophic-matrix domains were also up-regulated. Extracellular matrix proteins, proteases/protease inhibitors, and members of the Osiris gene family were prevalent among differentially regulated genes. However, analysis of Drosophila homologs of differentially expressed genes revealed spatial and temporal patterns consistent with developmental asynchrony as a likely confounder of our results. We therefore used qPCR to measure the consistency of gene expression changes for a subset of differentially expressed genes. A replicate experiment sampled at both 48 and 72 hours post infection allowed further discrimination of genes likely to be involved in host response. The consistently responsive genes in our test set included a hymenopteran-specific protein tyrosine kinase, a hymenopteran specific serine endopeptidase, a cytochrome P450 (CYP9Q1), and a homolog of trynity, a zona pellucida domain protein. Of the known honey bee antimicrobial peptides, apidaecin was responsive at both time-points studied whereas hymenoptaecin was more consistent in its level of change between biological replicates and had the greatest increase in expression by RNA-seq analysis.

Modeling on-column reduction of trisulfide bonds in monoclonal antibodies during protein A chromatography.

PubMed

Ghose, Sanchayita; Rajshekaran, Rupshika; Labanca, Marisa; Conley, Lynn

2017-01-06

Trisulfides can be a common post-translational modification in many recombinant monoclonal antibodies. These are a source of product heterogeneity that add to the complexity of product characterization and hence, need to be reduced for consistent product quality. Trisulfide bonds can be converted to the regular disulfide bonds by incorporating a novel cysteine wash step during Protein A affinity chromatography. An empirical model is developed for this on-column reduction reaction to compare the reaction rates as a function of typical operating parameters such as temperature, cysteine concentration, reaction time and starting level of trisulfides. The model presented here is anticipated to assist in the development of optimal wash conditions for the Protein A step to effectively reduce trisulfides to desired levels. Copyright © 2016 Elsevier B.V. All rights reserved.

Acute-phase proteins in relation to neuropsychiatric symptoms and use of psychotropic medication in Huntington's disease.

PubMed

Bouwens, J A; Hubers, A A M; van Duijn, E; Cobbaert, C M; Roos, R A C; van der Mast, R C; Giltay, E J

2014-08-01

Activation of the innate immune system has been postulated in the pathogenesis of Huntington's disease (HD). We studied serum concentrations of C-reactive protein (CRP) and low albumin as positive and negative acute-phase proteins in HD. Multivariate linear and logistic regression was used to study the association between acute-phase protein levels in relation to clinical, neuropsychiatric, cognitive, and psychotropic use characteristics in a cohort consisting of 122 HD mutation carriers and 42 controls at first biomarker measurement, and 85 HD mutation carriers and 32 controls at second biomarker measurement. Significant associations were found between acute-phase protein levels and Total Functioning Capacity (TFC) score, severity of apathy, cognitive impairment, and the use of antipsychotics. Interestingly, all significant results with neuropsychiatric symptoms disappeared after additional adjusting for antipsychotic use. High sensitivity CRP levels were highest and albumin levels were lowest in mutation carriers who continuously used antipsychotics during follow-up versus those that had never used antipsychotics (mean difference for CRP 1.4 SE mg/L; P=0.04; mean difference for albumin 3 SE g/L; P<0.001). The associations found between acute-phase proteins and TFC score, apathy, and cognitive impairment could mainly be attributed to the use of antipsychotics. This study provides evidence that HD mutation carriers who use antipsychotics are prone to develop an acute-phase response. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

Thyroid hormone affects secretory activity and uncoupling protein-3 expression in rat harderian gland.

PubMed

Chieffi Baccari, Gabriella; Monteforte, Rossella; de Lange, Pieter; Raucci, Franca; Farina, Paola; Lanni, Antonia

2004-07-01

The effects of T(3) administration on the rat Harderian gland were examined at morphological, biochemical, and molecular levels. T(3) induced hypertrophy of the two cell types (A and B) present in the glandular epithelium. In type A cells, the hypertrophy was mainly due to an increase in the size of the lipid compartment. The acinar lumina were filled with lipoproteic substances, and the cells often showed an olocrine secretory pattern. In type B cells, the hypertrophy largely consisted of a marked proliferation of mitochondria endowed with tightly packed cristae, the mitochondrial number being nearly doubled (from 62 to 101/100 microm(2)). Although the average area of individual mitochondria decreased by about 50%, the total area of the mitochondrial compartment increased by about 80% (from 11 to 19/100 microm(2)). This could be ascribed to T(3)-induced mitochondrial proliferation. The morphological and morphometric data correlated well with our biochemical results, which indicated that mitochondrial respiratory activity is increased in hyperthyroid rats. T(3), by influencing the metabolic function of the mitochondrial compartment, induces lipogenesis and the release of secretory product by type A cells. Mitochondrial uncoupling proteins 2 and 3 were expressed at both mRNA and protein levels in the euthyroid rat Harderian gland. T(3) treatment increased the mRNA levels of both uncoupling protein 2 (UCP2) and UCP3, but the protein level only of UCP3. A possible role for these proteins in the Harderian gland is discussed.

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

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

2014-10-01

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 individuals suggest that these SNPs also influence regulation of these proteins more generally and may therefore be relevant to other diseases.

Hypocholesterolaemic effects of lupin protein and pea protein/fibre combinations in moderately hypercholesterolaemic individuals.

PubMed

Sirtori, Cesare R; Triolo, Michela; Bosisio, Raffaella; Bondioli, Alighiero; Calabresi, Laura; De Vergori, Viviana; Gomaraschi, Monica; Mombelli, Giuliana; Pazzucconi, Franco; Zacherl, Christian; Arnoldi, Anna

2012-04-01

The present study was aimed to evaluate the effect of plant proteins (lupin protein or pea protein) and their combinations with soluble fibres (oat fibre or apple pectin) on plasma total and LDL-cholesterol levels. A randomised, double-blind, parallel group design was followed: after a 4-week run-in period, participants were randomised into seven treatment groups, each consisting of twenty-five participants. Each group consumed two bars containing specific protein/fibre combinations: the reference group consumed casein+cellulose; the second and third groups consumed bars containing lupin or pea proteins+cellulose; the fourth and fifth groups consumed bars containing casein and oat fibre or apple pectin; the sixth group and seventh group received bars containing combinations of pea protein and oat fibre or apple pectin, respectively. Bars containing lupin protein+cellulose ( - 116 mg/l, - 4·2%), casein+apple pectin ( - 152 mg/l, - 5·3%), pea protein+oat fibre ( - 135 mg/l, - 4·7%) or pea protein+apple pectin ( - 168 mg/l, - 6·4%) resulted in significant reductions of total cholesterol levels (P<0·05), whereas no cholesterol changes were observed in the subjects consuming the bars containing casein+cellulose, casein+oat fibre or pea protein+cellulose. The present study shows the hypocholesterolaemic activity and potential clinical benefits of consuming lupin protein or combinations of pea protein and a soluble fibre, such as oat fibre or apple pectin.

Cocoa and Whey Protein Differentially Affect Markers of Lipid and Glucose Metabolism and Satiety.

PubMed

Campbell, Caroline L; Foegeding, E Allen; Harris, G Keith

2016-03-01

Food formulation with bioactive ingredients is a potential strategy to promote satiety and weight management. Whey proteins are high in leucine and are shown to decrease hunger ratings and increase satiety hormone levels; cocoa polyphenolics moderate glucose levels and slow digestion. This study examined the effects of cocoa and whey proteins on lipid and glucose metabolism and satiety in vitro and in a clinical trial. In vitro, 3T3-L1 preadipocytes were treated with 0.5-100 μg/mL cocoa polyphenolic extract (CPE) and/or 1-15 mM leucine (Leu) and assayed for lipid accumulation and leptin production. In vivo, a 6-week clinical trial consisted of nine panelists (age: 22.6 ± 1.7; BMI: 22.3 ± 2.1) consuming chocolate-protein beverages once per week, including placebo, whey protein isolate (WPI), low polyphenolic cocoa (LP), high polyphenolic cocoa (HP), LP-WPI, and HP-WPI. Measurements included blood glucose and adiponectin levels, and hunger ratings at baseline and 0.5-4.0 h following beverage consumption. At levels of 50 and 100 μg/mL, CPE significantly inhibited preadipocyte lipid accumulation by 35% and 50%, respectively, and by 22% and 36% when combined with 15 mM Leu. Leu treatment increased adipocyte leptin production by 26-37%. In the clinical trial, all beverages significantly moderated blood glucose levels 30 min postconsumption. WPI beverages elicited lowest peak glucose levels and HP levels were significantly lower than LP. The WPI and HP beverage treatments significantly increased adiponectin levels, but elicited no significant changes in hunger ratings. These trends suggest that combinations of WPI and cocoa polyphenols may improve markers of metabolic syndrome and satiety.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warren, J.E.; Klaine, S.J.

The field cricket, Acheta domesticus, was used as a test organism to determine the effects of heavy metal exposure on cellular immunity. Insects were separated by sex and exposed to cadmium chloride or mercuric chloride at concentrations of 0, 2.5, and 5.0 ug/g. Exposures consisted of injecting the chemicals into the hemocoel of each insect on days 0, 2, and 4. Hemolymph was collected on day 7 of the study to determine total hemocyte counts, protein levels, and phenoloxidase activity in individual insects. Cadmium chloride decreased the total number of hemocytes in male crickets at 2.5 and 5.0 ug/g andmore » in female crickets at 5.0 ug/g. Protein levels increased in a dose dependent manner in the males but only slightly increased in the females. Mercuric chloride caused a dose-dependent increase in total hemocytes in both male and female crickets. In addition, mercuric chloride caused a dose-dependent increase in protein levels in males but not females.« less

Tutorial on Protein Ontology Resources

PubMed Central

Arighi, Cecilia; Drabkin, Harold; Christie, Karen R.; Ross, Karen; Natale, Darren

2017-01-01

The Protein Ontology (PRO) is the reference ontology for proteins in the Open Biomedical Ontologies (OBO) foundry and consists of three sub-ontologies representing protein classes of homologous genes, proteoforms (e.g., splice isoforms, sequence variants, and post-translationally modified forms), and protein complexes. PRO defines classes of proteins and protein complexes, both species-specific and species non-specific, and indicates their relationships in a hierarchical framework, supporting accurate protein annotation at the appropriate level of granularity, analyses of protein conservation across species, and semantic reasoning. In this first section of this chapter, we describe the PRO framework including categories of PRO terms and the relationship of PRO to other ontologies and protein resources. Next, we provide a tutorial about the PRO website (proconsortium.org) where users can browse and search the PRO hierarchy, view reports on individual PRO terms, and visualize relationships among PRO terms in a hierarchical table view, a multiple sequence alignment view, and a Cytoscape network view. Finally, we describe several examples illustrating the unique and rich information available in PRO. PMID:28150233

Statistical Model to Analyze Quantitative Proteomics Data Obtained by 18O/16O Labeling and Linear Ion Trap Mass Spectrometry

PubMed Central

Jorge, Inmaculada; Navarro, Pedro; Martínez-Acedo, Pablo; Núñez, Estefanía; Serrano, Horacio; Alfranca, Arántzazu; Redondo, Juan Miguel; Vázquez, Jesús

2009-01-01

Statistical models for the analysis of protein expression changes by stable isotope labeling are still poorly developed, particularly for data obtained by 16O/18O labeling. Besides large scale test experiments to validate the null hypothesis are lacking. Although the study of mechanisms underlying biological actions promoted by vascular endothelial growth factor (VEGF) on endothelial cells is of considerable interest, quantitative proteomics studies on this subject are scarce and have been performed after exposing cells to the factor for long periods of time. In this work we present the largest quantitative proteomics study to date on the short term effects of VEGF on human umbilical vein endothelial cells by 18O/16O labeling. Current statistical models based on normality and variance homogeneity were found unsuitable to describe the null hypothesis in a large scale test experiment performed on these cells, producing false expression changes. A random effects model was developed including four different sources of variance at the spectrum-fitting, scan, peptide, and protein levels. With the new model the number of outliers at scan and peptide levels was negligible in three large scale experiments, and only one false protein expression change was observed in the test experiment among more than 1000 proteins. The new model allowed the detection of significant protein expression changes upon VEGF stimulation for 4 and 8 h. The consistency of the changes observed at 4 h was confirmed by a replica at a smaller scale and further validated by Western blot analysis of some proteins. Most of the observed changes have not been described previously and are consistent with a pattern of protein expression that dynamically changes over time following the evolution of the angiogenic response. With this statistical model the 18O labeling approach emerges as a very promising and robust alternative to perform quantitative proteomics studies at a depth of several thousand proteins. PMID:19181660

Dietary n-6 PUFA, carbohydrate:protein ratio and change in body weight and waist circumference: a follow-up study.

PubMed

Jakobsen, Marianne U; Madsen, Lise; Dethlefsen, Claus; Due, Karen M; Halkjær, Jytte; Sørensen, Thorkild I A; Kristiansen, Karsten; Overvad, Kim

2015-05-01

To investigate the association between the intake of n-6 PUFA and subsequent change in body weight and waist circumference at different levels of the carbohydrate:protein ratio. Follow-up study with anthropometric measurements at recruitment and on average 5·3 years later. Dietary intake was determined at recruitment by using an FFQ that was designed for the study and validated. We applied linear regression models with 5-year change in weight or waist circumference as outcome and including a two-way interaction term between n-6 PUFA and carbohydrate intakes, lower-order terms, protein intake, long-chain n-3 PUFA intake and other potential confounders. Due to adjustment for intake of protein, levels of carbohydrate indirectly reflect levels of the carbohydrate:protein ratio. Diet, Cancer and Health follow-up study, Denmark. Women and men (n 29 152) aged 55 years. For a high intake of n-6 PUFA (6·9 % of energy) v. a low intake of n-6 PUFA (3·4 % of energy), the difference in 5-year weight change was -189·7 g (95 % CI -636·8, 257·4 g) at a low carbohydrate:protein ratio and -86·7 g (95 % CI -502·9, 329·6 g) at a high carbohydrate:protein ratio; the differences in 5-year waist circumference change were 0·26 cm (95 % CI -0·47, 0·98 cm) and -0·52 cm (95 % CI -1·19, 0·15 cm), respectively. Inclusion of the dietary glycaemic index did not change the results. No consistent associations between the intake of n-6 PUFA and change in body weight or waist circumference at different levels of the carbohydrate:protein ratio were observed.

Molecular modeling and SPRi investigations of interleukin 6 (IL6) protein and DNA aptamers.

PubMed

Rhinehardt, Kristen L; Vance, Stephen A; Mohan, Ram V; Sandros, Marinella; Srinivas, Goundla

2018-06-01

Interleukin 6 (IL6), an inflammatory response protein has major implications in immune-related inflammatory diseases. Identification of aptamers for the IL6 protein aids in diagnostic, therapeutic, and theranostic applications. Three different DNA aptamers and their interactions with IL6 protein were extensively investigated in a phosphate buffed saline (PBS) solution. Molecular-level modeling through molecular dynamics provided insights of structural, conformational changes and specific binding domains of these protein-aptamer complexes. Multiple simulations reveal consistent binding region for all protein-aptamer complexes. Conformational changes coupled with quantitative analysis of center of mass (COM) distance, radius of gyration (R g ), and number of intermolecular hydrogen bonds in each IL6 protein-aptamer complex was used to determine their binding performance strength and obtain molecular configurations with strong binding. A similarity comparison of the molecular configurations with strong binding from molecular-level modeling concurred with Surface Plasmon Resonance imaging (SPRi) for these three aptamer complexes, thus corroborating molecular modeling analysis findings. Insights from the natural progression of IL6 protein-aptamer binding modeled in this work has identified key features such as the orientation and location of the aptamer in the binding event. These key features are not readily feasible from wet lab experiments and impact the efficacy of the aptamers in diagnostic and theranostic applications.

Function, dynamics and evolution of network motif modules in integrated gene regulatory networks of worm and plant.

PubMed

Defoort, Jonas; Van de Peer, Yves; Vermeirssen, Vanessa

2018-06-05

Gene regulatory networks (GRNs) consist of different molecular interactions that closely work together to establish proper gene expression in time and space. Especially in higher eukaryotes, many questions remain on how these interactions collectively coordinate gene regulation. We study high quality GRNs consisting of undirected protein-protein, genetic and homologous interactions, and directed protein-DNA, regulatory and miRNA-mRNA interactions in the worm Caenorhabditis elegans and the plant Arabidopsis thaliana. Our data-integration framework integrates interactions in composite network motifs, clusters these in biologically relevant, higher-order topological network motif modules, overlays these with gene expression profiles and discovers novel connections between modules and regulators. Similar modules exist in the integrated GRNs of worm and plant. We show how experimental or computational methodologies underlying a certain data type impact network topology. Through phylogenetic decomposition, we found that proteins of worm and plant tend to functionally interact with proteins of a similar age, while at the regulatory level TFs favor same age, but also older target genes. Despite some influence of the duplication mode difference, we also observe at the motif and module level for both species a preference for age homogeneity for undirected and age heterogeneity for directed interactions. This leads to a model where novel genes are added together to the GRNs in a specific biological functional context, regulated by one or more TFs that also target older genes in the GRNs. Overall, we detected topological, functional and evolutionary properties of GRNs that are potentially universal in all species.

Serum S100B Represents a New Biomarker for Mood Disorders

PubMed Central

Schroeter, Matthias L.; Sacher, Julia; Steiner, Johann; Schoenknecht, Peter; Mueller, Karsten

2013-01-01

Recently, mood disorders have been discussed to be characterized by glial pathology. The protein S100B, a growth and differentiation factor, is located in, and may actively be released by astro- and oligodendrocytes. This protein is easily assessed in human serum and provides a useful parameter for glial activation or injury. Here, we review studies investigating the glial marker S100B in serum of patients with mood disorders. Studies consistently show that S100B is elevated in mood disorders; more strongly in major depressive than bipolar disorder. Consistent with the glial hypothesis of mood disorders, serum S100B levels interact with age with higher levels in elderly depressed subjects. Successful antidepressive treatment has been associated with serum S100B reduction in major depression, whereas there is no evidence of treatment effects in mania. In contrast to the glial marker S100B, the neuronal marker protein neuron-specific enolase is unaltered in mood disorders. Recently, serum S100B has been linked to specific imaging parameters in the human white matter suggesting a role for S100B as an oligodendrocytic marker protein. In sum, serum S100B can be regarded as a promising in vivo biomarker for mood disorders deepening the understanding of the pathogenesis and plasticity-changes in these disorders. Future longitudinal studies combining serum S100B with other cell-specific serum parameters and multimodal imaging are warranted to further explore this serum protein in the development, monitoring and treatment of mood disorders. PMID:23701298

Quantitative iTRAQ LC-MS/MS proteomics reveals metabolic responses to biofuel ethanol in cyanobacterial Synechocystis sp. PCC 6803.

PubMed

Qiao, Jianjun; Wang, Jiangxin; Chen, Lei; Tian, Xiaoxu; Huang, Siqiang; Ren, Xiaoyue; Zhang, Weiwen

2012-11-02

Recent progress in metabolic engineering has led to autotrophic production of ethanol in various cyanobacterial hosts. However, cyanobacteria are known to be sensitive to ethanol, which restricts further efforts to increase ethanol production levels in these renewable host systems. To understand the mechanisms of ethanol tolerance so that engineering more robust cyanobacterial hosts can be possible, in this study, the responses of model cyanobacterial Synechocystis sp. PCC 6803 to ethanol were determined using a quantitative proteomics approach with iTRAQ LC-MS/MS technologies. The resulting high-quality proteomic data set consisted of 24,887 unique peptides corresponding to 1509 identified proteins, a coverage of approximately 42% of the predicted proteins in the Synechocystis genome. Using a cutoff of 1.5-fold change and a p-value less than 0.05, 135 and 293 unique proteins with differential abundance levels were identified between control and ethanol-treated samples at 24 and 48 h, respectively. Functional analysis showed that the Synechocystis cells employed a combination of induced common stress response, modifications of cell membrane and envelope, and induction of multiple transporters and cell mobility-related proteins as protection mechanisms against ethanol toxicity. Interestingly, our proteomic analysis revealed that proteins related to multiple aspects of photosynthesis were up-regulated in the ethanol-treated Synechocystis cells, consistent with increased chlorophyll a concentration in the cells upon ethanol exposure. The study provided the first comprehensive view of the complicated molecular mechanisms against ethanol stress and also provided a list of potential gene targets for further engineering ethanol tolerance in Synechocystis PCC 6803.

The mechanism and properties of bio-photon emission and absorption in protein molecules in living systems

NASA Astrophysics Data System (ADS)

Pang, Xiao-feng

2012-05-01

The mechanism and properties of bio-photon emission and absorption in bio-tissues were studied using Pang's theory of bio-energy transport, in which the energy spectra of protein molecules are obtained from the discrete dynamic equation. From the energy spectra, it was determined that the protein molecules could both radiate and absorb bio-photons with wavelengths of <3 μm and 5-7 μm, consistent with the energy level transitions of the excitons. These results were consistent with the experimental data; this consisted of infrared absorption data from collagen, bovine serum albumin, the protein-like molecule acetanilide, plasma, and a person's finger, and the laser-Raman spectra of acidity I-type collagen in the lungs of a mouse, and metabolically active Escherichia coli. We further elucidated the mechanism responsible for the non-thermal biological effects produced by the infrared light absorbed by the bio-tissues, using the above results. No temperature rise was observed; instead, the absorbed infrared light promoted the vibrations of amides as well the transport of the bio-energy from one place to other in the protein molecules, which changed their conformations. These experimental results, therefore, not only confirmed the validity of the mechanism of bio-photon emission, and the newly developed theory of bio-energy transport mentioned above, but also explained the mechanism and properties of the non-thermal biological effects produced by the absorption of infrared light by the living systems.

FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eyre, Nicholas S.; Cleland, Leslie G.; Mayrhofer, Graham

2008-06-06

Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. Wemore » propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells.« less

Rapid calculation of accurate atomic charges for proteins via the electronegativity equalization method.

PubMed

Ionescu, Crina-Maria; Geidl, Stanislav; Svobodová Vařeková, Radka; Koča, Jaroslav

2013-10-28

We focused on the parametrization and evaluation of empirical models for fast and accurate calculation of conformationally dependent atomic charges in proteins. The models were based on the electronegativity equalization method (EEM), and the parametrization procedure was tailored to proteins. We used large protein fragments as reference structures and fitted the EEM model parameters using atomic charges computed by three population analyses (Mulliken, Natural, iterative Hirshfeld), at the Hartree-Fock level with two basis sets (6-31G*, 6-31G**) and in two environments (gas phase, implicit solvation). We parametrized and successfully validated 24 EEM models. When tested on insulin and ubiquitin, all models reproduced quantum mechanics level charges well and were consistent with respect to population analysis and basis set. Specifically, the models showed on average a correlation of 0.961, RMSD 0.097 e, and average absolute error per atom 0.072 e. The EEM models can be used with the freely available EEM implementation EEM_SOLVER.

S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death.

PubMed

Kohr, Mark J; Evangelista, Alicia M; Ferlito, Marcella; Steenbergen, Charles; Murphy, Elizabeth

2014-04-01

Oxidative stress and membrane damage following myocardial ischemia/reperfusion injury are important contributors to cardiomyocyte death and the loss of myocardial function. Our previous study identified cysteine 144 (C144) of tripartite motif-containing protein 72 (TRIM72) as a potential site for S-nitrosylation (SNO). TRIM72 is a cardioprotective membrane repair protein that can be both activated and targeted for degradation by different oxidative modifications. Consistent with the potential regulation of TRIM72 by various oxidative modifications, we found that SNO levels increased at C144 of TRIM72 with ischemic preconditioning. Therefore, to investigate the role of C144 in the regulation of TRIM72 function, we mutated C144 of TRIM72 to a serine residue (TRIM72(C144S)), and expressed either TRIM72(WT) or TRIM72(C144S) in HEK-293 cells, which lack endogenous TRIM72, in order to examine the effect of this mutation on the functional stability of TRIM72 and on cell survival. We hypothesized that SNO of TRIM72 stabilizes the protein, thus allowing for membrane repair and enhanced cell survival. Upon treatment with hydrogen peroxide (H2O2), we found that TRIM72(WT) levels were decreased, but not TRIM72(C144S) and this correlated with increased H2O2-induced cell death in TRIM72(WT) cells. Additionally, we found that treatment with the cardioprotective S-nitrosylating agent S-nitrosoglutathione (GSNO), was able to preserve TRIM72(WT) protein levels and enhance TRIM72(WT)-mediated cell survival, but had no effect on TRIM72(C144S) levels. Consistent with our hypothesis, GSNO was also found to increase SNO levels and inhibit H2O2-induced irreversible oxidation for TRIM72(WT) without affecting TRIM72(C144S). In further support of our hypothesis, GSNO blocked the ischemia/reperfusion-induced decrease in TRIM72 levels and reduced infarct size in a Langendorff-perfused heart model. The results of these studies have important implications for cardioprotection and suggest that SNO of TRIM72 at C144 prevents the oxidation-induced degradation of TRIM72 following oxidative insult, therefore enhancing cardiomyocyte survival. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular Dynamics Simulation of WSK-3, a Computationally Designed, Water-Soluble Variant of the Integral Membrane Protein KcsA

PubMed Central

Bronson, Jonathan; Lee, One-Sun; Saven, Jeffery G.

2006-01-01

Poor solubility and low expression levels often make membrane proteins difficult to study. An alternative to the use of detergents to solubilize these aggregation-prone proteins is the partial redesign of the sequence so as to confer water solubility. Recently, computationally assisted membrane protein solubilization (CAMPS) has been reported, where exposed hydrophobic residues on a protein's surface are computationally redesigned. Herein, the structure and fluctuations of a designed, water-soluble variant of KcsA (WSK-3) were studied using molecular dynamics simulations. The root mean square deviation of the protein from its starting structure, where the backbone coordinates are those of KcsA, was 1.8 Å. The structure of salt bridges involved in structural specificity and solubility were examined. The preferred configuration of ions and water in the selectivity filter of WSK-3 was consistent with the reported preferences for KcsA. The structure of the selectivity filter was maintained, which is consistent with WSK-3 having an affinity for agitoxin2 comparable to that of wild-type KcsA. In contrast to KcsA, the central cavity's side chains were observed to reorient, allowing water diffusion through the side of the cavity wall. These simulations provide an atomistic analysis of the CAMPS strategy and its implications for further investigations of membrane proteins. PMID:16299086

Efficient lowering of triglyceride levels in mice by human apoAV protein variants associated with hypertriglyceridemia.

PubMed

Vaessen, Stefan F C; Sierts, Jeroen A; Kuivenhoven, Jan Albert; Schaap, Frank G

2009-02-06

Variation in the apolipoprotein A5 (APOA5) gene has consistently been associated with increased plasma triglyceride (TG) levels in epidemiological studies. In vivo functionality of these variations, however, has thus far not been tested. Using adenoviral over-expression, we evaluated plasma expression levels and TG-lowering efficacies of wild-type human apoAV, two human apoAV variants associated with increased TG (S19W, G185C) and one variant (Q341H) that is predicted to have altered protein function. Injection of mice with adenovirus encoding wild-type or mutant apoAV resulted in an identical dose-dependent elevation of human apoAV levels in plasma. The increase in apoAV levels resulted in pronounced lowering of plasma TG levels at two viral dosages. Unexpectedly, the TG-lowering efficacy of all three apoAV variants was similar to wild-type apoAV. In addition, no effect on TG-hydrolysis-related plasma parameters (free fatty acids, glycerol and post-heparin lipoprotein lipase activity) was apparent upon expression of all apoAV variants. In conclusion, our data indicate that despite their association with hypertriglyceridemia and/or predicted protein dysfunction, the 19W, 185C and 341H apoAV variants are equally effective in reducing plasma TG levels in mice.

Comparative proteomic and physiological analyses reveal the protective effect of exogenous polyamines in the bermudagrass (Cynodon dactylon) response to salt and drought stresses.

PubMed

Shi, Haitao; Ye, Tiantian; Chan, Zhulong

2013-11-01

Polyamines conferred enhanced abiotic stress tolerance in multiple plant species. However, the effect of polyamines on abiotic stress and physiological change in bermudagrass, the most widely used warm-season turfgrasses, are unknown. In this study, pretreatment of exogenous polyamine conferred increased salt and drought tolerances in bermudagrass. Comparative proteomic analysis was performed to further investigate polyamines mediated responses, and 36 commonly regulated proteins by at least two types of polyamines in bermudagrass were successfully identified, including 12 proteins with increased level, 20 proteins with decreased level and other 4 specifically expressed proteins. Among them, proteins involved in electron transport and energy pathways were largely enriched, and nucleoside diphosphate kinase (NDPK) and three antioxidant enzymes were extensively regulated by polyamines. Dissection of reactive oxygen species (ROS) levels indicated that polyamine-derived H2O2 production might play dual roles under abiotic stress conditions. Moreover, accumulation of osmolytes was also observed after application of exogenous polyamines, which is consistent with proteomics results that several proteins involved in carbon fixation pathway were mediated commonly by polyamines pretreatment. Taken together, we proposed that polyamines could activate multiple pathways that enhance bermudagrass adaption to salt and drought stresses. These findings might be applicable for genetically engineering of grasses and crops to improve stress tolerance.

Cottonseed protein derivatives as nutritional and functional supplements in food formulations.

PubMed

Cherry, J P; Berardi, L C; Zarins, Z M; Wadsworth, J I; Vinnett, C H

1978-01-01

Cottonseeds contain protein with desirable food functional and nutritional properties. Storage globulins make up most of the protein stored in cottonseed and can be separated into five fractions by gel filtration chromatography. Each fraction is distinguishable from the other by its amino acid and polyacrylamide gel electrophoretic properties. Proteins of cottonseed contribute greatly to the functional properties of emulsions, co-isolates, and texturized derivatives. For example, increasing the amount of high protein cottonseed flour in wheat suspensions from 2% to 10% improved the capacity (54-97 ml of oil) and viscosity (5,000-100,000+ cps) of emulsions. The 10% suspension formed emulsions with increasing oil capacity (84-100 ml) and viscosity (28,000-100,000+ cps) as the pH was adjusted from 4.5 to 9.5. Consistencies of the products ranged from that of salad dressing (low percent suspensions, or acid pH) to that of mayonnaise (high percent, or basic pH). These data were utilized to derive a multiple regression model to predict optimum use of cottonseed proteins in emulsions of varying consistencies. A coprecipitated isolate containing greater than 94% protein was prepared from a blend of cottonseed and peanut flours. Amino acid content of the co-isolate reflected that of the protein in the two flours of the composite. The co-isolate has lower gossypol level and improved color and functional properties than a cottonseed protein isolate. Storage protein isolate of cottonseed suspended in aqueous solution and heated with constant stirring forms a texturized product; the quality of the product depends on heat, pH, salt, and the quantity of nonstorage proteins. Protein and amino acid content of meat products were improved by the addition of the texturized protein of cottonseed.

Matrix metalloproteinase-9, a potential biological marker in invasive pituitary adenomas.

PubMed

Gong, Jian; Zhao, Yunge; Abdel-Fattah, Rana; Amos, Samson; Xiao, Aizhen; Lopes, M Beatriz S; Hussaini, Isa M; Laws, Edward R

2008-01-01

We analyzed MMP-9 expression using mRNA and protein level determinations and explored the possibility that matrix metalloproteinase-9 (MMP-9) is a potential biological marker of pituitary adenoma invasiveness and whether MMP-9 could be used to discriminate the extent of invasiveness among different hormonal subtypes, tumor sizes, growth characteristics, and primary versus recurrent tumors. 73 pituitary tumor specimens were snap frozen in liquid nitrogen immediately after surgical resection. RNA and protein were extracted. MMP-9 mRNA transcripts were analyzed by quantitative RT-PCR. MMP-9 protein activity was analyzed by gelatin zymography and validated by western blot analysis. Immunohistochemistry was performed to identify the presence and localization of MMP-9 in pituitary adenomas. Statistical differences between results were determined using Student's t-test or one way ANOVA. Comparing different hormonal subtypes of noninvasive and invasive pituitary tumors, MMP-9 mRNA expression was significantly increased in the majority of invasive adenomas. Considering the protein levels, our data also showed a significant increase in MMP-9 activity in the majority of invasive adenomas and these differences were confirmed by western blot analysis and immunohistochemistry. In addition, consistent differences in MMP-9 expression levels were found according to tumor subtype, tumor size, tumor extension and primary versus redo-surgery. MMP-9 expression can consistently distinguish invasive pituitary tumors from noninvasive pituitary tumors and would reflect the extent of invasiveness in pituitary tumors according to tumor subtype, size, tumor extension, primary and redo surgery, even at early stages of invasiveness. MMP-9 may be considered a potential biomarker to determine and predict the invasive nature of pituitary tumors.

Expression of holo and apo forms of spinach acyl carrier protein-I in leaves of transgenic tobacco plants.

PubMed Central

Post-Beittenmiller, M A; Schmid, K M; Ohlrogge, J B

1989-01-01

Acyl carrier protein (ACP) is a chloroplast-localized cofactor of fatty acid synthesis, desaturation, and acyl transfer. We have transformed tobacco with a chimeric gene consisting of the tobacco ribulose-1,5-bisphosphate carboxylase promoter and transit peptide and the sequence encoding the mature spinach ACP-I. Spinach ACP-I was expressed in the transformed plants at levels twofold to threefold higher than the endogenous tobacco ACPs as determined by protein immunoblots and assays of ACP in leaf extracts. In addition to these elevated levels of the holo form, there were high levels of apoACP-I, a form lacking the 4'-phosphopantetheine prosthetic group and not previously detected in vivo. The mature forms of both apoACP-I and holoACP-I were located in the chloroplasts, indicating that the transit peptide was cleaved and that attachment of the prosthetic group was not required for uptake into the plastid. There were also significant levels of spinach acyl-ACP-I, demonstrating that spinach ACP-I participated in tobacco fatty acid metabolism. Lipid analyses of the transformed plants indicated that the increased ACP levels caused no significant alterations in leaf lipid biosynthesis. PMID:2535529

Differential quantitative proteomics of Porphyromonas gingivalis by linear ion trap mass spectrometry: non-label methods comparison, q-values and LOWESS curve fitting

PubMed Central

Xia, Qiangwei; Wang, Tiansong; Park, Yoonsuk; Lamont, Richard J.; Hackett, Murray

2009-01-01

Differential analysis of whole cell proteomes by mass spectrometry has largely been applied using various forms of stable isotope labeling. While metabolic stable isotope labeling has been the method of choice, it is often not possible to apply such an approach. Four different label free ways of calculating expression ratios in a classic “two-state” experiment are compared: signal intensity at the peptide level, signal intensity at the protein level, spectral counting at the peptide level, and spectral counting at the protein level. The quantitative data were mined from a dataset of 1245 qualitatively identified proteins, about 56% of the protein encoding open reading frames from Porphyromonas gingivalis, a Gram-negative intracellular pathogen being studied under extracellular and intracellular conditions. Two different control populations were compared against P. gingivalis internalized within a model human target cell line. The q-value statistic, a measure of false discovery rate previously applied to transcription microarrays, was applied to proteomics data. For spectral counting, the most logically consistent estimate of random error came from applying the locally weighted scatter plot smoothing procedure (LOWESS) to the most extreme ratios generated from a control technical replicate, thus setting upper and lower bounds for the region of experimentally observed random error. PMID:19337574

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

PubMed

Bykowski, Tomasz; Babb, Kelly; von Lackum, Kate; Riley, Sean P; Norris, Steven J; Stevenson, Brian

2006-07-01

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

Examination of the relation between periodontal health status and cardiovascular risk factors: serum total and high density lipoprotein cholesterol, C-reactive protein, and plasma fibrinogen.

PubMed

Wu, T; Trevisan, M; Genco, R J; Falkner, K L; Dorn, J P; Sempos, C T

2000-02-01

Using data from the Third National Health and Nutrition Examination Survey (1988-1994), the authors examined the relation between periodontal health and cardiovascular risk factors: serum total and high density lipoprotein cholesterol, C-reactive protein, and plasma fibrinogen. A total of 10,146 participants were included in the analyses of cholesterol and C-reactive protein and 4,461 in the analyses of fibrinogen. Periodontal health indicators included the gingival bleeding index, calculus index, and periodontal disease status (defined by pocket depth and attachment loss). While cholesterol and fibrinogen were analyzed as continuous variables, C-reactive protein was dichotomized into two levels. The results show a significant relation between indicators of poor periodontal status and increased C-reactive protein and fibrinogen. The association between periodontal status and total cholesterol level is much weaker. No consistent association between periodontal status and high density lipoprotein cholesterol was detectable. Similar patterns of association were observed for participants aged 17-54 years and those 55 years and older. In conclusion, this study suggests that total cholesterol, C-reactive protein, and fibrinogen are possible intermediate factors that may link periodontal disease to elevated cardiovascular risk.

G protein-coupled odorant receptors: From sequence to structure

PubMed Central

de March, Claire A; Kim, Soo-Kyung; Antonczak, Serge; Goddard, William A; Golebiowski, Jérôme

2015-01-01

Odorant receptors (ORs) are the largest subfamily within class A G protein-coupled receptors (GPCRs). No experimental structural data of any OR is available to date and atomic-level insights are likely to be obtained by means of molecular modeling. In this article, we critically align sequences of ORs with those GPCRs for which a structure is available. Here, an alignment consistent with available site-directed mutagenesis data on various ORs is proposed. Using this alignment, the choice of the template is deemed rather minor for identifying residues that constitute the wall of the binding cavity or those involved in G protein recognition. PMID:26044705

Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

PubMed Central

Ferrero, Rut; Torres, Magdalena

2002-01-01

Background Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability. PMID:12350235

Expression of BMI-1 and Mel-18 in breast tissue - a diagnostic marker in patients with breast cancer

PubMed Central

2010-01-01

Background Polycomb Group (PcG) proteins are epigenetic silencers involved in maintaining cellular identity, and their deregulation can result in cancer. Expression of Mel-18 and Bmi-1 has been studied in tumor tissue, but not in adjacent non-cancerous breast epithelium. Our study compares the expression of the two genes in normal breast epithelium of cancer patients and relates it to the level of expression in the corresponding tumors as well as in breast epithelium of healthy women. Methods A total of 79 tumors, of which 71 malignant tumors of the breast, 6 fibroadenomas, and 2 DCIS were studied and compared to the reduction mammoplastic specimens of 11 healthy women. In addition there was available adjacent cancer free tissue for 23 of the malignant tumors. The tissue samples were stored in RNAlater, RNA was isolated to create expression microarray profile. These two genes were then studied more closely first on mRNA transcription level by microarrays (Agilent 44 K) and quantitative RT-PCR (TaqMan) and then on protein expression level using immunohistochemistry. Results Bmi-1 mRNA is significantly up-regulated in adjacent normal breast tissue in breast cancer patients compared to normal breast tissue from noncancerous patients. Conversely, mRNA transcription level of Mel-18 is lower in normal breast from patients operated for breast cancer compared to breast tissue from mammoplasty. When protein expression of these two genes was evaluated, we observed that most of the epithelial cells were positive for Bmi-1 in both groups of tissue samples, although the expression intensity was stronger in normal tissue from cancer patients compared to mammoplasty tissue samples. Protein expression of Mel-18 showed inversely stronger intensity in tissue samples from mammoplasty compared to normal breast tissue from patients operated for breast cancer. Conclusion Bmi-1 mRNA level is consistently increased and Mel-18 mRNA level is consistently decreased in adjacent normal breast tissue of cancer patients as compared to normal breast tissue in women having had reduction mammoplasties. Bmi-1/Mel-18 ratio can be potentially used as a tool for stratifying women at risk of developing malignancy. PMID:21162745

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

PubMed Central

Halter, David; Collart, Martine A.; Panasenko, Olesya O.

2014-01-01

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

Machine Learning-based Classification of Diffuse Large B-cell Lymphoma Patients by Their Protein Expression Profiles.

PubMed

Deeb, Sally J; Tyanova, Stefka; Hummel, Michael; Schmidt-Supprian, Marc; Cox, Juergen; Mann, Matthias

2015-11-01

Characterization of tumors at the molecular level has improved our knowledge of cancer causation and progression. Proteomic analysis of their signaling pathways promises to enhance our understanding of cancer aberrations at the functional level, but this requires accurate and robust tools. Here, we develop a state of the art quantitative mass spectrometric pipeline to characterize formalin-fixed paraffin-embedded tissues of patients with closely related subtypes of diffuse large B-cell lymphoma. We combined a super-SILAC approach with label-free quantification (hybrid LFQ) to address situations where the protein is absent in the super-SILAC standard but present in the patient samples. Shotgun proteomic analysis on a quadrupole Orbitrap quantified almost 9,000 tumor proteins in 20 patients. The quantitative accuracy of our approach allowed the segregation of diffuse large B-cell lymphoma patients according to their cell of origin using both their global protein expression patterns and the 55-protein signature obtained previously from patient-derived cell lines (Deeb, S. J., D'Souza, R. C., Cox, J., Schmidt-Supprian, M., and Mann, M. (2012) Mol. Cell. Proteomics 11, 77-89). Expression levels of individual segregation-driving proteins as well as categories such as extracellular matrix proteins behaved consistently with known trends between the subtypes. We used machine learning (support vector machines) to extract candidate proteins with the highest segregating power. A panel of four proteins (PALD1, MME, TNFAIP8, and TBC1D4) is predicted to classify patients with low error rates. Highly ranked proteins from the support vector analysis revealed differential expression of core signaling molecules between the subtypes, elucidating aspects of their pathobiology. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Antisense oligonucleotide against GSK-3β in brain of SAMP8 mice improves learning and memory and decreases oxidative stress: Involvement of transcription factor Nrf2 and implications for Alzheimer disease.

PubMed

Farr, Susan A; Ripley, Jessica L; Sultana, Rukhsana; Zhang, Zhaoshu; Niehoff, Michael L; Platt, Thomas L; Murphy, M Paul; Morley, John E; Kumar, Vijaya; Butterfield, D Allan

2014-02-01

Glycogen synthase kinase (GSK)-3β is a multifunctional protein that has been implicated in the pathological characteristics of Alzheimer's disease (AD), including the heightened levels of neurofibrillary tangles, amyloid-beta (Aβ), and neurodegeneration. In this study we used 12-month-old SAMP8 mice, an AD model, to examine the effects GSK-3β may cause regarding the cognitive impairment and oxidative stress associated with AD. To suppress the level of GSK-3β, SAMP8 mice were treated with an antisense oligonucleotide (GAO) directed at this kinase. We measured a decreased level of GSK-3β in the cortex of the mice, indicating the success of the antisense treatment. Learning and memory assessments of the SAMP8 mice were tested post-antisense treatment using an aversive T-maze and object recognition test, both of which observably improved. In cortex samples of the SAMP8 mice, decreased levels of protein carbonyl and protein-bound HNE were measured, indicating decreased oxidative stress. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a transcription factor known to increase the level of many antioxidants, including glutathione-S transferase (GST), and is negatively regulated by the activity of GSK-3β. Our results indicated the increased nuclear localization of Nrf2 and level of GST, suggesting the increased activity of the transcription factor as a result of GSK-3β suppression, consistent with the decreased oxidative stress observed. Consistent with the improved learning and memory, and consistent with GSK-3b being a tau kinase, we observed decreased tau phosphorylation in brain of GAO-treated SAMP8 mice compared to that of RAO-treated SAMP8 mice. Lastly, we examined the ability of GAO to cross the blood-brain barrier and determined it to be possible. The results presented in this study demonstrate that reducing GSK-3 with a phosphorothionated antisense against GSK-3 improves learning and memory, reduces oxidative stress, possibly coincident with increased levels of the antioxidant transcriptional activity of Nrf2, and decreases tau phosphorylation. Our study supports the notion of GAO as a possible treatment for AD. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

Use of 13Cα Chemical-Shifts in Protein Structure Determination

PubMed Central

Vila, Jorge A.; Ripoll, Daniel R.; Scheraga, Harold A.

2008-01-01

A physics-based method, aimed at determining protein structures by using NOE-derived distances together with observed and computed 13C chemical shifts, is proposed. The approach makes use of 13Cα chemical shifts, computed at the density functional level of theory, to obtain torsional constraints for all backbone and side-chain torsional angles without making a priori use of the occupancy of any region of the Ramachandran map by the amino acid residues. The torsional constraints are not fixed but are changed dynamically in each step of the procedure, following an iterative self-consistent approach intended to identify a set of conformations for which the computed 13Cα chemical shifts match the experimental ones. A test is carried out on a 76-amino acid all-α-helical protein, namely the B. Subtilis acyl carrier protein. It is shown that, starting from randomly generated conformations, the final protein models are more accurate than an existing NMR-derived structure model of this protein, in terms of both the agreement between predicted and observed 13Cα chemical shifts and some stereochemical quality indicators, and of similar accuracy as one of the protein models solved at a high level of resolution. The results provide evidence that this methodology can be used not only for structure determination but also for additional protein structure refinement of NMR-derived models deposited in the Protein Data Bank. PMID:17516673

AMMOS2: a web server for protein-ligand-water complexes refinement via molecular mechanics.

PubMed

Labbé, Céline M; Pencheva, Tania; Jereva, Dessislava; Desvillechabrol, Dimitri; Becot, Jérôme; Villoutreix, Bruno O; Pajeva, Ilza; Miteva, Maria A

2017-07-03

AMMOS2 is an interactive web server for efficient computational refinement of protein-small organic molecule complexes. The AMMOS2 protocol employs atomic-level energy minimization of a large number of experimental or modeled protein-ligand complexes. The web server is based on the previously developed standalone software AMMOS (Automatic Molecular Mechanics Optimization for in silico Screening). AMMOS utilizes the physics-based force field AMMP sp4 and performs optimization of protein-ligand interactions at five levels of flexibility of the protein receptor. The new version 2 of AMMOS implemented in the AMMOS2 web server allows the users to include explicit water molecules and individual metal ions in the protein-ligand complexes during minimization. The web server provides comprehensive analysis of computed energies and interactive visualization of refined protein-ligand complexes. The ligands are ranked by the minimized binding energies allowing the users to perform additional analysis for drug discovery or chemical biology projects. The web server has been extensively tested on 21 diverse protein-ligand complexes. AMMOS2 minimization shows consistent improvement over the initial complex structures in terms of minimized protein-ligand binding energies and water positions optimization. The AMMOS2 web server is freely available without any registration requirement at the URL: http://drugmod.rpbs.univ-paris-diderot.fr/ammosHome.php. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Comparative proteome analysis of Aspergillus oryzae 3.042 and A. oryzae 100-8 strains: Towards the production of different soy sauce flavors.

PubMed

Zhao, Guozhong; Hou, Lihua; Yao, Yunping; Wang, Chunling; Cao, Xiaohong

2012-07-16

Aspergillus oryzae plays a central role in soybean fermentation, particularly in its contribution to the flavor of soy sauce. We present a comparative assessment of the intracellular differences between wild-type strain 3.042 and mutant strain A100-8, at the proteome level. 522 different protein spots were identified by MALDI-TOF MS, with 134 spots being confirmed by MALDI-TOF MS/MS. Of these, 451 were differentially expressed proteins (DEPs). There was at least a two-fold increase for 288 spots, and at least a two-fold decrease for 163 spots, in strain A100-8 when compared to 3.042. Further analysis showed that 63 of the more abundant proteins were involved in glycolysis and the citrate cycle; 43 more abundant proteins and 10 less abundant proteins were related to amino acid biosynthesis and metabolism; two of the more abundant proteins were involved in vitamin biosynthesis; and five of the more abundant proteins and four of the less abundant proteins were related to secondary metabolites. Moreover, quantitative real time PCR showed that the mRNA expression levels of six typical genes we selected were consistent with changes in protein expression. We postulate that there may be a relationship between DEPs and the flavor formation mechanism in A. oryzae. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Protein sequence annotation in the genome era: the annotation concept of SWISS-PROT+TREMBL.

PubMed

Apweiler, R; Gateau, A; Contrino, S; Martin, M J; Junker, V; O'Donovan, C; Lang, F; Mitaritonna, N; Kappus, S; Bairoch, A

1997-01-01

SWISS-PROT is a curated protein sequence database which strives to provide a high level of annotation, a minimal level of redundancy and high level of integration with other databases. Ongoing genome sequencing projects have dramatically increased the number of protein sequences to be incorporated into SWISS-PROT. Since we do not want to dilute the quality standards of SWISS-PROT by incorporating sequences without proper sequence analysis and annotation, we cannot speed up the incorporation of new incoming data indefinitely. However, as we also want to make the sequences available as fast as possible, we introduced TREMBL (TRanslation of EMBL nucleotide sequence database), a supplement to SWISS-PROT. TREMBL consists of computer-annotated entries in SWISS-PROT format derived from the translation of all coding sequences (CDS) in the EMBL nucleotide sequence database, except for CDS already included in SWISS-PROT. While TREMBL is already of immense value, its computer-generated annotation does not match the quality of SWISS-PROTs. The main difference is in the protein functional information attached to sequences. With this in mind, we are dedicating substantial effort to develop and apply computer methods to enhance the functional information attached to TREMBL entries.

Loss of retinoschisin (RS1) cell surface protein in maturing mouse rod photoreceptors elevates the luminance threshold for light-driven translocation of transducin but not arrestin.

PubMed

Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bush, Ronald A; Sieving, Paul A

2012-09-19

Loss of retinoschisin (RS1) in Rs1 knock-out (Rs1-KO) retina produces a post-photoreceptor phenotype similar to X-linked retinoschisis in young males. However, Rs1 is expressed strongly in photoreceptors, and Rs1-KO mice have early reduction in the electroretinogram a-wave. We examined light-activated transducin and arrestin translocation in young Rs1-KO mice as a marker for functional abnormalities in maturing rod photoreceptors. We found a progressive reduction in luminance threshold for transducin translocation in wild-type (WT) retinas between postnatal days P18 and P60. At P21, the threshold in Rs1-KO retinas was 10-fold higher than WT, but it decreased to <2.5-fold higher by P60. Light-activated arrestin translocation and re-translocation of transducin in the dark were not affected. Rs1-KO rod outer segment (ROS) length was significantly shorter than WT at P21 but was comparable with WT at P60. These findings suggested a delay in the structural and functional maturation of Rs1-KO ROS. Consistent with this, transcription factors CRX and NRL, which are fundamental to maturation of rod protein expression, were reduced in ROS of Rs1-KO mice at P21 but not at P60. Expression of transducin was 15-30% lower in P21 Rs1-KO ROS and transducin GTPase hydrolysis was nearly twofold faster, reflecting a 1.7- to 2.5-fold increase in RGS9 (regulator of G-protein signaling) level. Transduction protein expression and activity levels were similar to WT at P60. Transducin translocation threshold elevation indicates photoreceptor functional abnormalities in young Rs1-KO mice. Rapid reduction in threshold coupled with age-related changes in transduction protein levels and transcription factor expression are consistent with delayed maturation of Rs1-KO photoreceptors.

An observational study of sequential protein-sparing, very low-calorie ketogenic diet (Oloproteic diet) and hypocaloric Mediterranean-like diet for the treatment of obesity.

PubMed

Castaldo, Giuseppe; Monaco, Luigi; Castaldo, Laura; Galdo, Giovanna; Cereda, Emanuele

2016-09-01

The impact of a rehabilitative multi-step dietary program consisting in different diets has been scantily investigated. In an open-label study, 73 obese patients underwent a two-phase weight loss (WL) program: a 3-week protein-sparing, very low-calorie, ketogenic diet (<500 kcal/day; Oloproteic(®) Diet) and a 6-week hypocaloric (25-30 kcal/kg of ideal body weight/day), low glycemic index, Mediterranean-like diet (hypo-MD). Both phases improved visceral adiposity, liver enzymes, GH levels, blood pressure and glucose and lipid metabolism. However, the hypo-MD was responsible for a re-increase in blood lipids and glucose tolerance parameters. Changes in visceral adiposity and glucose control-related variables were more consistent in patients with metabolic syndrome. However, in these patients the hypo-MD did not result in a consistent re-increase in glucose control-related variables. A dietary program consisting in a ketogenic regimen followed by a balanced MD appeared to be feasible and efficacious in reducing cardiovascular risk, particularly in patients with metabolic syndrome.

Lactoferrin binding protein B – a bi-functional bacterial receptor protein

PubMed Central

Ostan, Nicholas K. H.; Yu, Rong-Hua; Ng, Dixon; Lai, Christine Chieh-Lin; Sarpe, Vladimir; Schriemer, David C.

2017-01-01

Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB), there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation. PMID:28257520

Inhibition of transforming growth factor-beta1-induced signaling and epithelial-to-mesenchymal transition by the Smad-binding peptide aptamer Trx-SARA.

PubMed

Zhao, Bryan M; Hoffmann, F Michael

2006-09-01

Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor beta (TGF-beta) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and beta-catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, Trx-SARA, which comprises a rigid scaffold, the Escherichia coli thioredoxin A protein (Trx), displaying a constrained 56-amino acid Smad-binding motif from the Smad anchor for receptor activation (SARA) protein. Trx-SARA bound specifically to Smad2 and Smad3 and inhibited both TGF-beta-induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-SARA had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-beta1. Trx-SARA was primarily localized to the nucleus and perturbed the normal cytoplasmic localization of Smad2 and 3 to a nuclear localization in the absence of TGF-beta1, consistent with reduced Smad nuclear export. The key mode of action of Trx-SARA was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-beta1 stimulation, a mechanism of action consistent with the preferential binding of SARA to monomeric Smad protein and Trx-SARA-mediated disruption of active Smad complexes.

Retinal expression of Wnt-pathway mediated genes in low-density lipoprotein receptor-related protein 5 (Lrp5) knockout mice.

PubMed

Chen, Jing; Stahl, Andreas; Krah, Nathan M; Seaward, Molly R; Joyal, Jean-Sebastian; Juan, Aimee M; Hatton, Colman J; Aderman, Christopher M; Dennison, Roberta J; Willett, Keirnan L; Sapieha, Przemyslaw; Smith, Lois E H

2012-01-01

Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5) impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. The defective retinal vasculature phenotype in human FEVR patients is recapitulated in Lrp5 knockout (Lrp5(-/-)) mouse with delayed and incomplete development of retinal vessels. In this study we examined gene expression changes in the developing Lrp5(-/-) mouse retina to gain insight into the molecular mechanisms that underlie the pathology of FEVR in humans. Gene expression levels were assessed with an Illumina microarray on total RNA from Lrp5(-/-) and WT retinas isolated on postnatal day (P) 8. Regulated genes were confirmed using RT-qPCR analysis. Consistent with a role in vascular development, we identified expression changes in genes involved in cell-cell adhesion, blood vessel morphogenesis and membrane transport in Lrp5(-/-) retina compared to WT retina. In particular, tight junction protein claudin5 and amino acid transporter slc38a5 are both highly down-regulated in Lrp5(-/-) retina. Similarly, several Wnt ligands including Wnt7b show decreased expression levels. Plasmalemma vesicle associated protein (plvap), an endothelial permeability marker, in contrast, is up-regulated consistent with increased permeability in Lrp5(-/-) retinas. Together these data suggest that Lrp5 regulates multiple groups of genes that influence retinal angiogenesis and may contribute to the pathogenesis of FEVR.

Impact of different wort boiling temperatures on the beer foam stabilizing properties of lipid transfer protein 1.

PubMed

Van Nierop, Sandra N E; Evans, David E; Axcell, Barry C; Cantrell, Ian C; Rautenbach, Marina

2004-05-19

Beer consumers demand satisfactory and consistent foam stability; thus, it is a high priority for brewers. Beer foam is stabilized by the interaction between certain beer proteins, including lipid transfer protein 1 (LTP1), and isomerized hop alpha-acids, but destabilized by lipids. In this study it was shown that the wort boiling temperature during the brewing process was critical in determining the final beer LTP1 content and conformation. LTP1 levels during brewing were measured by an LTP1 ELISA, using antinative barley LTP1 polyclonal antibodies. It was observed that the higher wort boiling temperatures ( approximately 102 degrees C), resulting from low altitude at sea level, reduced the final beer LTP1 level to 2-3 microg/mL, whereas the lower wort boiling temperatures ( approximately 96 degrees C), resulting from higher altitudes (1800 m), produced LTP1 levels between 17 and 35 microg/mL. Low levels of LTP1 in combination with elevated levels of free fatty acids (FFA) resulted in poor foam stability, whereas beer produced with low levels of LTP1 and FFA had satisfactory foam stability. Previous studies indicated the need for LTP1 denaturing to improve its foam stabilizing properties. However, the results presented here show that LTP1 denaturation reduces its ability to act as a binding protein for foam-damaging FFA. These investigations suggest that wort boiling temperature is an important factor in determining the level and conformation of LTP1, thereby favoring satisfactory beer foam stability.

Characterization of DNA-protein interactions using high-throughput sequencing data from pulldown experiments

NASA Astrophysics Data System (ADS)

Moreland, Blythe; Oman, Kenji; Curfman, John; Yan, Pearlly; Bundschuh, Ralf

Methyl-binding domain (MBD) protein pulldown experiments have been a valuable tool in measuring the levels of methylated CpG dinucleotides. Due to the frequent use of this technique, high-throughput sequencing data sets are available that allow a detailed quantitative characterization of the underlying interaction between methylated DNA and MBD proteins. Analyzing such data sets, we first found that two such proteins cannot bind closer to each other than 2 bp, consistent with structural models of the DNA-protein interaction. Second, the large amount of sequencing data allowed us to find rather weak but nevertheless clearly statistically significant sequence preferences for several bases around the required CpG. These results demonstrate that pulldown sequencing is a high-precision tool in characterizing DNA-protein interactions. This material is based upon work supported by the National Science Foundation under Grant No. DMR-1410172.

Light-induced protein degradation in human-derived cells.

PubMed

Sun, Wansheng; Zhang, Wenyao; Zhang, Chao; Mao, Miaowei; Zhao, Yuzheng; Chen, Xianjun; Yang, Yi

2017-05-27

Controlling protein degradation can be a valuable tool for posttranslational regulation of protein abundance to study complex biological systems. In the present study, we designed a light-switchable degron consisting of a light oxygen voltage (LOV) domain of Avena sativa phototropin 1 (AsLOV2) and a C-terminal degron. Our results showed that the light-switchable degron could be used for rapid and specific induction of protein degradation in HEK293 cells by light in a proteasome-dependent manner. Further studies showed that the light-switchable degron could also be utilized to mediate the degradation of secreted Gaussia princeps luciferase (GLuc), demonstrating the adaptability of the light-switchable degron in different types of protein. We suggest that the light-switchable degron offers a robust tool to control protein levels and may serves as a new and significant method for gene- and cell-based therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

The high mobility group AT-hook 1 protein stimulates bovine herpesvirus 1 productive infection.

PubMed

Zhu, Liqian; Jones, Clinton

2017-06-15

Bovine herpesvirus 1 (BoHV-1) is an important pathogen of cattle that causes clinical symptoms in the upper respiratory tract and conjunctivitis. Like most alpha-herpesvirinae subfamily members, BoHV-1 establishes latency in sensory neurons. Stress consistently induces reactivation from latency, which is essential for virus transmission. Recent studies demonstrated that a viral protein (ORF2) expressed in a subset of latently infected neurons is associated with β-catenin and the high mobility group AT-hook 1 protein (HMGA1), which correlates with increased expression of these proteins in latently infected neurons. Since HMGA1 is primarily expressed in actively growing cells, binds to the minor groove of A+T rich regions in double-stranded DNA, and mediates gene transcription, we hypothesized that HMGA1 regulates BoHV-1 productive infection. Studies in this report indicated that productive infection increased HMGA1 protein levels and re-localized the protein in the nucleus. Netropsin, a small molecule that binds to the minor groove of DNA and prevents HMGA1 from interacting with DNA inhibited viral replication and interfered with the ability of BoHV-1 to induce HMGA1 re-localization. Furthermore, netropsin reduced RNA and protein expression of two viral regulatory proteins (bICP0 and bICP22) during productive infection, but increased bICP4 levels. Small interfering RNAs (siRNAs) that specifically target HMGA1 reduced HMGA1 RNA levels and virus production confirming HMGA1 stimulates productive infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Periodontal and serum protein profiles in patients with rheumatoid arthritis treated with tumor necrosis factor inhibitor adalimumab.

PubMed

Kobayashi, Tetsuo; Yokoyama, Tomoko; Ito, Satoshi; Kobayashi, Daisuke; Yamagata, Akira; Okada, Moe; Oofusa, Ken; Narita, Ichiei; Murasawa, Akira; Nakazono, Kiyoshi; Yoshie, Hiromasa

2014-11-01

Tumor necrosis factor (TNF)-α inhibitor has been shown to affect the periodontal condition of patients with rheumatoid arthritis (RA). The aim of the present study is to assess the effect of a fully humanized anti-TNF-α monoclonal antibody, adalimumab (ADA), on the periodontal condition of patients with RA and to compare serum protein profiles before and after ADA therapy. The study participants consisted of 20 patients with RA treated with ADA. Clinical periodontal and rheumatologic parameters and serum cytokine levels were evaluated at baseline and 3 months later. Serum protein spot volume was examined with two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with significant difference in abundance before and after ADA therapy were found and identified using mass spectrometry and protein databases. The patients showed a significant decrease in gingival index (P = 0.002), bleeding on probing (P = 0.003), probing depth (P = 0.002), disease activity score including 28 joints using C-reactive protein (P <0.001), and serum levels of TNF-α (P <0.001) and interleukin-6 (P <0.001) after ADA medication, although plaque levels were comparable. Among a total of 495 protein spots obtained, nine spots were significantly decreased in abundance at reassessment, corresponding to complement factor H, phospholipase D, serum amyloid A, complement component 4, and α-1-acid glycoprotein (P <0.01). These results suggest a beneficial effect of ADA therapy on the periodontal condition of patients with RA, which might be related to differences in serum protein profiles before and after ADA therapy.

Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raymond, Amy; Lovell, Scott; Lorimer, Don

2009-12-01

With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. colimore » and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.« less

The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA*

PubMed Central

Wigington, Callie P.; Morris, Kevin J.; Newman, Laura E.; Corbett, Anita H.

2016-01-01

Polyadenosine RNA-binding proteins (Pabs) regulate multiple steps in gene expression. This protein family includes the well studied Pabs, PABPN1 and PABPC1, as well as the newly characterized Pab, zinc finger CCCH-type containing protein 14 (ZC3H14). Mutations in ZC3H14 are linked to a form of intellectual disability. To probe the function of ZC3H14, we performed a transcriptome-wide analysis of cells depleted of either ZC3H14 or the control Pab, PABPN1. Depletion of PABPN1 affected ∼17% of expressed transcripts, whereas ZC3H14 affected only ∼1% of expressed transcripts. To assess the function of ZC3H14 in modulating target mRNAs, we selected the gene encoding the ATP synthase F0 subunit C (ATP5G1) transcript. Knockdown of ZC3H14 significantly reduced ATP5G1 steady-state mRNA levels. Consistent with results suggesting that ATP5G1 turnover increases upon depletion of ZC3H14, double knockdown of ZC3H14 and the nonsense-mediated decay factor, UPF1, rescues ATP5G1 transcript levels. Furthermore, fractionation reveals an increase in the amount of ATP5G1 pre-mRNA that reaches the cytoplasm when ZC3H14 is depleted and that ZC3H14 binds to ATP5G1 pre-mRNA in the nucleus. These data support a role for ZC3H14 in ensuring proper nuclear processing and retention of ATP5G1 pre-mRNA. Consistent with the observation that ATP5G1 is a rate-limiting component for ATP synthase activity, knockdown of ZC3H14 decreases cellular ATP levels and causes mitochondrial fragmentation. These data suggest that ZC3H14 modulates pre-mRNA processing of select mRNA transcripts and plays a critical role in regulating cellular energy levels, observations that have broad implications for proper neuronal function. PMID:27563065

Morphine Regulated Synaptic Networks Revealed by Integrated Proteomics and Network Analysis*

PubMed Central

Stockton, Steven D.; Gomes, Ivone; Liu, Tong; Moraje, Chandrakala; Hipólito, Lucia; Jones, Matthew R.; Ma'ayan, Avi; Morón, Jose A.; Li, Hong; Devi, Lakshmi A.

2015-01-01

Despite its efficacy, the use of morphine for the treatment of chronic pain remains limited because of the rapid development of tolerance, dependence and ultimately addiction. These undesired effects are thought to be because of alterations in synaptic transmission and neuroplasticity within the reward circuitry including the striatum. In this study we used subcellular fractionation and quantitative proteomics combined with computational approaches to investigate the morphine-induced protein profile changes at the striatal postsynaptic density. Over 2,600 proteins were identified by mass spectrometry analysis of subcellular fractions enriched in postsynaptic density associated proteins from saline or morphine-treated striata. Among these, the levels of 34 proteins were differentially altered in response to morphine. These include proteins involved in G-protein coupled receptor signaling, regulation of transcription and translation, chaperones, and protein degradation pathways. The altered expression levels of several of these proteins was validated by Western blotting analysis. Using Genes2Fans software suite we connected the differentially expressed proteins with proteins identified within the known background protein-protein interaction network. This led to the generation of a network consisting of 116 proteins with 40 significant intermediates. To validate this, we confirmed the presence of three proteins predicted to be significant intermediates: caspase-3, receptor-interacting serine/threonine protein kinase 3 and NEDD4 (an E3-ubiquitin ligase identified as a neural precursor cell expressed developmentally down-regulated protein 4). Because this morphine-regulated network predicted alterations in proteasomal degradation, we examined the global ubiquitination state of postsynaptic density proteins and found it to be substantially altered. Together, these findings suggest a role for protein degradation and for the ubiquitin/proteasomal system in the etiology of opiate dependence and addiction. PMID:26149443

A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

PubMed Central

Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

1996-01-01

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 Fig. 7 Fig. 8 PMID:8632983

Differential effects of nicotine treatment and ethanol self-administration on CYP2A6, CYP2B6 and nicotine pharmacokinetics in African green monkeys.

PubMed

Ferguson, C S; Miksys, S; Palmour, R M; Tyndale, R F

2012-12-01

In primates, nicotine is metabolically inactivated in the liver by CYP2A6 and possibly CYP2B6. Changes in the levels of these two enzymes may affect nicotine pharmacokinetics and influence smoking behaviors. This study investigated the independent and combined effects of ethanol self-administration and nicotine treatment (0.5 mg/kg b.i.d. s.c.) on hepatic CYP2A6 and CYP2B6 levels (mRNA, protein, and enzymatic activity), in vitro nicotine metabolism, and in vivo nicotine pharmacokinetics in monkeys. CYP2A6 mRNA and protein levels and in vitro coumarin (selective CYP2A6 substrate) and nicotine metabolism were decreased by nicotine treatment but unaffected by ethanol. CYP2B6 protein levels and in vitro bupropion (selective CYP2B6 substrate) metabolism were increased by ethanol but unaffected by nicotine treatment; CYP2B6 mRNA levels were unaltered by either treatment. Combined ethanol and nicotine exposure decreased CYP2A6 mRNA and protein levels, as well as in vitro coumarin and nicotine metabolism, and increased CYP2B6 protein levels and in vitro bupropion metabolism, with no change in CYP2B6 mRNA levels. Chronic nicotine resulted in higher nicotine plasma levels achieved after nicotine administration, consistent with decreased CYP2A6. Ethanol alone, or combined with nicotine, resulted in lower nicotine plasma levels by a mechanism independent of the change in these enzymes. Thus, nicotine can decrease hepatic CYP2A6, reducing the metabolism of its substrates, including nicotine, whereas ethanol can increase hepatic CYP2B6, increasing the metabolism of CYP2B6 substrates. In vivo nicotine pharmacokinetics are differentially affected by ethanol and nicotine, but when both drugs are used in combination the effect more closely resembles ethanol alone.

Application of clustering methods: Regularized Markov clustering (R-MCL) for analyzing dengue virus similarity

NASA Astrophysics Data System (ADS)

Lestari, D.; Raharjo, D.; Bustamam, A.; Abdillah, B.; Widhianto, W.

2017-07-01

Dengue virus consists of 10 different constituent proteins and are classified into 4 major serotypes (DEN 1 - DEN 4). This study was designed to perform clustering against 30 protein sequences of dengue virus taken from Virus Pathogen Database and Analysis Resource (VIPR) using Regularized Markov Clustering (R-MCL) algorithm and then we analyze the result. By using Python program 3.4, R-MCL algorithm produces 8 clusters with more than one centroid in several clusters. The number of centroid shows the density level of interaction. Protein interactions that are connected in a tissue, form a complex protein that serves as a specific biological process unit. The analysis of result shows the R-MCL clustering produces clusters of dengue virus family based on the similarity role of their constituent protein, regardless of serotypes.

PPP2R1A regulated by PAX3/FOXO1 fusion contributes to the acquisition of aggressive behavior in PAX3/FOXO1-positive alveolar rhabdomyosarcoma.

PubMed

Akaike, Keisuke; Suehara, Yoshiyuki; Kohsaka, Shinji; Hayashi, Takuo; Tanabe, Yu; Kazuno, Saiko; Mukaihara, Kenta; Toda-Ishii, Midori; Kurihara, Taisei; Kim, Youngji; Okubo, Taketo; Hayashi, Yasuhide; Takamochi, Kazuya; Takahashi, Fumiyuki; Kaneko, Kazuo; Ladanyi, Marc; Saito, Tsuyoshi

2018-05-18

To better characterize the oncogenic role of the PAX3-FOXO1 fusion protein in the acquisition of aggressive behavior in ARMS, we employed a proteomic approach using a PAX3-FOXO1 knockdown system in ARMS cell lines. This approach revealed a protein list consisting of 107 consistently upregulated and 114 consistently downregulated proteins that were expected to be regulated by PAX3-FOXO1 fusion protein. Furthermore, we identified 16 upregulated and 17 downregulated critical proteins based on a data-mining analysis. We also evaluated the function of PPP2R1A in ARMS cells. The PPP2R1A expression was upregulated at both the mRNA and protein levels by PAX3-FOXO1 silencing. The silencing of PPP2R1A significantly increased the cell growth of all four ARMS cells, suggesting that PPP2R1A still has a tumor suppressive function in ARMS cells; however, the native expression of PPP2R1A was low in the presence of PAX3-FOXO1. In addition, the activation of PP2A-part of which was encoded by PPP2R1A -by FTY720 treatment in ARMS cell lines inhibited cell growth. On the human phospho-kinase array analysis of 46 specific Ser/Thr or Tyr phosphorylation sites on 39 selected proteins, eNOS, AKT1/2/3, RSK1/2/3 and STAT3 phosphorylation were decreased by FTY-720 treatment. These findings suggest that PPP2R1A is a negatively regulated by PAX3-FOXO1 in ARMS. The activation of PP2A-probably in combination with kinase inhibitors-may represent a therapeutic target in ARMS. We believe that the protein expression profile associated with PAX3-FOXO1 would be valuable for discovering new therapeutic targets in ARMS.

An ensemble approach to protein fold classification by integration of template-based assignment and support vector machine classifier.

PubMed

Xia, Jiaqi; Peng, Zhenling; Qi, Dawei; Mu, Hongbo; Yang, Jianyi

2017-03-15

Protein fold classification is a critical step in protein structure prediction. There are two possible ways to classify protein folds. One is through template-based fold assignment and the other is ab-initio prediction using machine learning algorithms. Combination of both solutions to improve the prediction accuracy was never explored before. We developed two algorithms, HH-fold and SVM-fold for protein fold classification. HH-fold is a template-based fold assignment algorithm using the HHsearch program. SVM-fold is a support vector machine-based ab-initio classification algorithm, in which a comprehensive set of features are extracted from three complementary sequence profiles. These two algorithms are then combined, resulting to the ensemble approach TA-fold. We performed a comprehensive assessment for the proposed methods by comparing with ab-initio methods and template-based threading methods on six benchmark datasets. An accuracy of 0.799 was achieved by TA-fold on the DD dataset that consists of proteins from 27 folds. This represents improvement of 5.4-11.7% over ab-initio methods. After updating this dataset to include more proteins in the same folds, the accuracy increased to 0.971. In addition, TA-fold achieved >0.9 accuracy on a large dataset consisting of 6451 proteins from 184 folds. Experiments on the LE dataset show that TA-fold consistently outperforms other threading methods at the family, superfamily and fold levels. The success of TA-fold is attributed to the combination of template-based fold assignment and ab-initio classification using features from complementary sequence profiles that contain rich evolution information. http://yanglab.nankai.edu.cn/TA-fold/. yangjy@nankai.edu.cn or mhb-506@163.com. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Proteomic Analysis and qRT-PCR Verification of Temperature Response to Arthrospira (Spirulina) platensis

PubMed Central

Huili, Wang; Xiaokai, Zhao; Meili, Lin; Dahlgren, Randy A.; Wei, Chen; Jaiopeng, Zhou; Chengyang, Xu; Chunlei, Jin; Yi, Xu; Xuedong, Wang; Li, Ding; Qiyu, Bao

2013-01-01

Arthrospira (Spirulina) platensis (ASP) is a representative filamentous, non-N2-fixing cyanobacterium that has great potential to enhance the food supply and possesses several valuable physiological features. ASP tolerates high and low temperatures along with highly alkaline and salty environments, and can strongly resist oxidation and irradiation. Based on genomic sequencing of ASP, we compared the protein expression profiles of this organism under different temperature conditions (15°C, 35°Cand 45°C) using 2-DE and peptide mass fingerprinting techniques. A total of 122 proteins having a significant differential expression response to temperature were retrieved. Of the positively expressed proteins, the homologies of 116 ASP proteins were found in Arthrospira (81 proteins in Arthrospira platensis str. Paraca and 35 in Arthrospira maxima CS-328). The other 6 proteins have high homology with other microorganisms. We classified the 122 differentially expressed positive proteins into 14 functions using the COG database, and characterized their respective KEGG metabolism pathways. The results demonstrated that these differentially expressed proteins are mainly involved in post-translational modification (protein turnover, chaperones), energy metabolism (photosynthesis, respiratory electron transport), translation (ribosomal structure and biogenesis) and carbohydrate transport and metabolism. Others proteins were related to amino acid transport and metabolism, cell envelope biogenesis, coenzyme metabolism and signal transduction mechanisms. Results implied that these proteins can perform predictable roles in rendering ASP resistance against low and high temperatures. Subsequently, we determined the transcription level of 38 genes in vivo in response to temperature and identified them by qRT-PCR. We found that the 26 differentially expressed proteins, representing 68.4% of the total target genes, maintained consistency between transcription and translation levels. The remaining 12 genes showed inconsistent protein expression with transcription level and accounted for 31.6% of the total target genes. PMID:24349519

2D-DIGE-based proteome expression changes in leaves of rice seedlings exposed to low-level gamma radiation at Iitate village, Fukushima

PubMed Central

Hayashi, Gohei; Moro, Carlo F; Rohila, Jai Singh; Shibato, Junko; Kubo, Akihiro; Imanaka, Tetsuji; Kimura, Shinzo; Ozawa, Shoji; Fukutani, Satoshi; Endo, Satoru; Ichikawa, Katsuki; Agrawal, Ganesh Kumar; Shioda, Seiji; Hori, Motohide; Fukumoto, Manabu; Rakwal, Randeep

2015-01-01

The present study continues our previous research on investigating the biological effects of low-level gamma radiation in rice at the heavily contaminated Iitate village in Fukushima, by extending the experiments to unraveling the leaf proteome. 14-days-old plants of Japonica rice (Oryza sativa L. cv. Nipponbare) were subjected to gamma radiation level of upto 4 µSv/h, for 72 h. Following exposure, leaf samples were taken from the around 190 µSv/3 d exposed seedling and total proteins were extracted. The gamma irradiated leaf and control leaf (harvested at the start of the experiment) protein lysates were used in a 2-D differential gel electrophoresis (2D-DIGE) experiment using CyDye labeling in order to asses which spots were differentially represented, a novelty of the study. 2D-DIGE analysis revealed 91 spots with significantly different expression between samples (60 positive, 31 negative). MALDI-TOF and TOF/TOF mass spectrometry analyses revealed those as comprising of 59 different proteins (50 up-accumulated, 9 down-accumulated). The identified proteins were subdivided into 10 categories, according to their biological function, which indicated that the majority of the differentially expressed proteins consisted of the general (non-energy) metabolism and stress response categories. Proteome-wide data point to some effects of low-level gamma radiation exposure on the metabolism of rice leaves. PMID:26451896

Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

PubMed Central

Kirkpatrick, Christopher; Maurer, Lisa M.; Oyelakin, Nikki E.; Yoncheva, Yuliya N.; Maurer, Russell; Slonczewski, Joan L.

2001-01-01

Acetate and formate are major fermentation products of Escherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-pta strain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of the ackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins. PMID:11591692

Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules▿ †

PubMed Central

Rahaman, Abdur; Miao, Wei; Turkewitz, Aaron P.

2009-01-01

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal β/γ-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, ΔGRT1 ΔGRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in ΔGRT1 ΔGRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from ΔGRT1 ΔGRT2 cells appear less adhesive than those from the wild type. PMID:19684282

Independent transport and sorting of functionally distinct protein families in Tetrahymena thermophila dense core secretory granules.

PubMed

Rahaman, Abdur; Miao, Wei; Turkewitz, Aaron P

2009-10-01

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal beta/gamma-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, DeltaGRT1 DeltaGRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in DeltaGRT1 DeltaGRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from DeltaGRT1 DeltaGRT2 cells appear less adhesive than those from the wild type.

Proteomic Analysis of Serum from Patients with Major Depressive Disorder to Compare Their Depressive and Remission Statuses

PubMed Central

Lee, Jiyeong; Joo, Eun-Jeong; Lim, Hee-Joung; Park, Jong-Moon; Lee, Kyu Young; Park, Arum; Seok, AeEun

2015-01-01

Objective Currently, there are a few biological markers to aid in the diagnosis and treatment of depression. However, it is not sufficient for diagnosis. We attempted to identify differentially expressed proteins during depressive moods as putative diagnostic biomarkers by using quantitative proteomic analysis of serum. Methods Blood samples were collected twice from five patients with major depressive disorder (MDD) at depressive status before treatment and at remission status during treatment. Samples were individually analyzed by liquid chromatography-tandem mass spectrometry for protein profiling. Differentially expressed proteins were analyzed by label-free quantification. Enzyme-linked immunosorbent assay (ELISA) results and receiver-operating characteristic (ROC) curves were used to validate the differentially expressed proteins. For validation, 8 patients with MDD including 3 additional patients and 8 matched normal controls were analyzed. Results The quantitative proteomic studies identified 10 proteins that were consistently upregulated or downregulated in 5 MDD patients. ELISA yielded results consistent with the proteomic analysis for 3 proteins. Expression levels were significantly different between normal controls and MDD patients. The 3 proteins were ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4 and complement component 1qC, which were upregulated during the depressive status. The depressive status could be distinguished from the euthymic status from the ROC curves for these proteins, and this discrimination was enhanced when all 3 proteins were analyzed together. Conclusion This is the first proteomic study in MDD patients to compare intra-individual differences dependent on mood. This technique could be a useful approach to identify MDD biomarkers, but requires additional proteomic studies for validation. PMID:25866527

An RNA electrophoretic mobility shift and mutational analysis of rnp-4f 5′-UTR intron splicing regulatory proteins in Drosophila reveals a novel new role for a dADAR protein isoform

PubMed Central

Lakshmi, G. Girija; Ghosh, Sushmita; Jones, Gabriel P.; Parikh, Roshni; Rawlins, Bridgette A.; Vaughn, Jack C.

2014-01-01

Alternative splicing greatly enhances the diversity of proteins encoded by eukaryotic genomes, and is also important in gene expression control. In contrast to the great depth of knowledge as to molecular mechanisms in the splicing pathway itself, relatively little is known about the regulatory events behind this process. The 5′-UTR and 3′-UTR in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation, and nearly 4,000 of the roughly 14,000 protein coding genes in Drosophila contain introns of unknown functional significance in their 5′-UTR. Here we report the results of an RNA electrophoretic mobility shift analysis of Drosophila rnp-4f 5′-UTR intron 0 splicing regulatory proteins. The pre-mRNA potential regulatory element consists of an evolutionarily-conserved 177-nt stem-loop arising from pairing of intron 0 with part of adjacent exon 2. Incubation of in vitro transcribed probe with embryo protein extract is shown to result in two shifted RNA-protein bands, and protein extract from a dADAR null mutant fly line results in only one shifted band. A mutated stem-loop in which the conserved exon 2 primary sequence is changed but secondary structure maintained by introducing compensatory base changes results in diminished band shifts. To test the hypothesis that dADAR plays a role in intron splicing regulation in vivo, levels of unspliced rnp-4f mRNA in dADAR mutant were compared to wild-type via real-time qRT-PCR. The results show that during embryogenesis unspliced rnp-4f mRNA levels fall by up to 85% in the mutant, in support of the hypothesis. Taken together, these results demonstrate a novel role for dADAR protein in rnp-4f 5′-UTR alternative intron splicing regulation which is consistent with a previously proposed model. PMID:23026215

Comprehensive analysis of gene expression patterns in Friedreich's ataxia fibroblasts by RNA sequencing reveals altered levels of protein synthesis factors and solute carriers

PubMed Central

Li, Yanjie; Lu, Yue; Lin, Kevin; Hauser, Lauren A.; Lynch, David R.

2017-01-01

ABSTRACT Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease usually caused by large homozygous expansions of GAA repeat sequences in intron 1 of the frataxin (FXN) gene. FRDA patients homozygous for GAA expansions have low FXN mRNA and protein levels when compared with heterozygous carriers or healthy controls. Frataxin is a mitochondrial protein involved in iron–sulfur cluster synthesis, and many FRDA phenotypes result from deficiencies in cellular metabolism due to lowered expression of FXN. Presently, there is no effective treatment for FRDA, and biomarkers to measure therapeutic trial outcomes and/or to gauge disease progression are lacking. Peripheral tissues, including blood cells, buccal cells and skin fibroblasts, can readily be isolated from FRDA patients and used to define molecular hallmarks of disease pathogenesis. For instance, FXN mRNA and protein levels as well as FXN GAA-repeat tract lengths are routinely determined using all of these cell types. However, because these tissues are not directly involved in disease pathogenesis, their relevance as models of the molecular aspects of the disease is yet to be decided. Herein, we conducted unbiased RNA sequencing to profile the transcriptomes of fibroblast cell lines derived from 18 FRDA patients and 17 unaffected control individuals. Bioinformatic analyses revealed significantly upregulated expression of genes encoding plasma membrane solute carrier proteins in FRDA fibroblasts. Conversely, the expression of genes encoding accessory factors and enzymes involved in cytoplasmic and mitochondrial protein synthesis was consistently decreased in FRDA fibroblasts. Finally, comparison of genes differentially expressed in FRDA fibroblasts to three previously published gene expression signatures defined for FRDA blood cells showed substantial overlap between the independent datasets, including correspondingly deficient expression of antioxidant defense genes. Together, these results indicate that gene expression profiling of cells derived from peripheral tissues can, in fact, consistently reveal novel molecular pathways of the disease. When performed on statistically meaningful sample group sizes, unbiased global profiling analyses utilizing peripheral tissues are critical for the discovery and validation of FRDA disease biomarkers. PMID:29125828

Perturbations in the apoptotic pathway and mitochondrial network dynamics in peripheral blood mononuclear cells from bipolar disorder patients

PubMed Central

Scaini, G; Fries, G R; Valvassori, S S; Zeni, C P; Zunta-Soares, G; Berk, M; Soares, J C; Quevedo, J

2017-01-01

Bipolar disorder (BD) is a severe psychiatric disorder characterized by phasic changes of mood and can be associated with progressive structural brain change and cognitive decline. The numbers and sizes of glia and neurons are reduced in several brain areas, suggesting the involvement of apoptosis in the pathophysiology of BD. Because the changes in mitochondrial dynamics are closely related with the early process of apoptosis and the specific processes of apoptosis and mitochondrial dynamics in BD have not been fully elucidated, we measured the apoptotic pathway and the expression of mitochondrial fission/fusion proteins from BD patients and healthy controls. We recruited 16 patients with BD type I and sixteen well-matched healthy controls and investigated protein levels of several pro-apoptotic and anti-apoptotic factors, as well as the expression of mitochondrial fission/fusion proteins in peripheral blood mononuclear cells (PBMCs). Our results showed that the levels of the anti-apoptotic proteins Bcl-xL, survivin and Bcl-xL/Bak dimer were significantly decreased, while active caspase-3 protein levels were significantly increased in PBMCs from BD patients. Moreover, we observed the downregulation of the mitochondrial fusion-related proteins Mfn2 and Opa1 and the upregulation of the fission protein Fis1 in PBMCs from BD patients, both in terms of gene expression and protein levels. We also showed a significantly decrease in the citrate synthase activity. Finally, we found a positive correlation between Mfn2 and Opa1 with mitochondrial content markers, as well as a negative correlation between mitochondrial fission/fusion proteins and apoptotic markers. Overall, data reported here are consistent with the working hypothesis that apoptosis may contribute to cellular dysfunction, brain volume loss and progressive cognitive in BD. Moreover, we show an important relationship between mitochondrial dynamics and the cell death pathway activation in BD patients, supporting the link between mitochondrial dysfunction and the pathophysiology of BD. PMID:28463235

3Drefine: an interactive web server for efficient protein structure refinement

PubMed Central

Bhattacharya, Debswapna; Nowotny, Jackson; Cao, Renzhi; Cheng, Jianlin

2016-01-01

3Drefine is an interactive web server for consistent and computationally efficient protein structure refinement with the capability to perform web-based statistical and visual analysis. The 3Drefine refinement protocol utilizes iterative optimization of hydrogen bonding network combined with atomic-level energy minimization on the optimized model using a composite physics and knowledge-based force fields for efficient protein structure refinement. The method has been extensively evaluated on blind CASP experiments as well as on large-scale and diverse benchmark datasets and exhibits consistent improvement over the initial structure in both global and local structural quality measures. The 3Drefine web server allows for convenient protein structure refinement through a text or file input submission, email notification, provided example submission and is freely available without any registration requirement. The server also provides comprehensive analysis of submissions through various energy and statistical feedback and interactive visualization of multiple refined models through the JSmol applet that is equipped with numerous protein model analysis tools. The web server has been extensively tested and used by many users. As a result, the 3Drefine web server conveniently provides a useful tool easily accessible to the community. The 3Drefine web server has been made publicly available at the URL: http://sysbio.rnet.missouri.edu/3Drefine/. PMID:27131371

The RNA Binding Protein CsrA Is a Pleiotropic Regulator of the Locus of Enterocyte Effacement Pathogenicity Island of Enteropathogenic Escherichia coli▿

PubMed Central

Bhatt, Shantanu; Edwards, Adrianne Nehrling; Nguyen, Hang Thi Thu; Merlin, Didier; Romeo, Tony; Kalman, Daniel

2009-01-01

The attaching and effacing (A/E) pathogen enteropathogenic Escherichia coli (EPEC) forms characteristic actin-filled membranous protrusions upon infection of host cells termed pedestals. Here we examine the role of the RNA binding protein CsrA in the expression of virulence genes and proteins that are necessary for pedestal formation. The csrA mutant was defective in forming actin pedestals on epithelial cells and in disrupting transepithelial resistance across polarized epithelial cells. Consistent with reduced pedestal formation, secretion of the translocators EspA, EspB, and EspD and the effector Tir was substantially reduced in the csrA mutant. Purified CsrA specifically bound to the sepL espADB mRNA leader, and the corresponding transcript levels were reduced in the csrA mutant. In contrast, Tir synthesis was unaffected in the csrA mutant. Reduced secretion of Tir appeared to be in part due to decreased synthesis of EscD, an inner membrane architectural protein of the type III secretion system (TTSS) and EscF, a protein that forms the protruding needle complex of the TTSS. These effects were not mediated through the locus of enterocyte effacement (LEE) transcriptional regulator GrlA or Ler. In contrast to the csrA mutant, multicopy expression of csrA repressed transcription from LEE1, grlRA, LEE2, LEE5, escD, and LEE4, an effect mediated by GrlA and Ler. Consistent with its role in other organisms, CsrA also regulated flagellar motility and glycogen levels. Our findings suggest that CsrA governs virulence factor expression in an A/E pathogen by regulating mRNAs encoding translocators, effectors, or transcription factors. PMID:19581394

The ras/mitogen-activated protein kinase pathway inhibitor and likely tumor suppressor proteins, sprouty 1 and sprouty 2 are deregulated in breast cancer.

PubMed

Lo, Ting Ling; Yusoff, Permeen; Fong, Chee Wai; Guo, Ke; McCaw, Ben J; Phillips, Wayne A; Yang, He; Wong, Esther Sook Miin; Leong, Hwei Fen; Zeng, Qi; Putti, Thomas Choudary; Guy, Graeme R

2004-09-01

Sprouty (Spry) proteins were found to be endogenous inhibitors of the Ras/mitogen-activated protein kinase pathway that play an important role in the remodeling of branching tissues. We investigated Spry expression levels in various cancers and found that Spry1 and Spry2 were down-regulated consistently in breast cancers. Such prevalent patterns of down-regulation may herald the later application of these isoforms as tumor markers that are breast cancer specific and more profound than currently characterized markers. Spry1 and 2 were expressed specifically in the luminal epithelial cells of breast ducts, with higher expression during stages of tissue remodeling when the epithelial ducts are forming and branching. These findings suggest that Sprys might be involved as a modeling counterbalance and surveillance against inappropriate epithelial expansion. The abrogation of endogenous Spry activity in MCF-7 cells by the overexpression of a previously characterized dominant-negative mutant of Spry, hSpry2Y55F resulted in enhanced cell proliferation in vitro. The hSpry2Y55F stably expressing cells also formed larger and greater number of colonies in the soft-agar assay. An in vivo nude mice assay showed a dramatic increase in the tumorigenic potential of hSpry2Y55F stable cells. The consistent down-regulation of Spry1 and 2 in breast cancer and the experimental evidence using a dominant-negative hSpry2Y55F indicate that Spry proteins may actively maintain tissue integrity that runs amok when their expression is decreased below normal threshold levels. This alludes to a previously unrecognized role for Sprys in cancer development.

Tumor microenvironment conditions alter Akt and Na+/H+ exchanger NHE1 expression in endothelial cells more than hypoxia alone: implications for endothelial cell function in cancer.

PubMed

Pedersen, A K; Mendes Lopes de Melo, J; Mørup, N; Tritsaris, K; Pedersen, S F

2017-08-14

Chronic angiogenesis is a hallmark of most tumors and takes place in a hostile tumor microenvironment (TME) characterized by hypoxia, low nutrient and glucose levels, elevated lactate and low pH. Despite this, most studies addressing angiogenic signaling use hypoxia as a proxy for tumor conditions. Here, we compared the effects of hypoxia and TME conditions on regulation of the Na + /H + exchanger NHE1, Ser/Thr kinases Akt1-3, and downstream effectors in endothelial cells. Human umbilical vein endothelial cells (HUVEC) and Ea.hy926 endothelial cells were exposed to simulated TME (1% hypoxia, low serum, glucose, pH, high lactate) or 1% hypoxia for 24 or 48 h, with or without NHE1 inhibition or siRNA-mediated knockdown. mRNA and protein levels of NHE1, Akt1-3, and downstream effectors were assessed by qPCR and Western blotting, vascular endothelial growth factor (VEGF) release by ELISA, and motility by scratch assay. Within 24 h, HIF-1α level and VEGF mRNA level were increased robustly by TME and modestly by hypoxia alone. The NHE1 mRNA level was decreased by both hypoxia and TME, and NHE1 protein was reduced by TME in Ea.hy926 cells. Akt1-3 mRNA was detected in HUVEC and Ea.hy926 cells, Akt1 most abundantly. Akt1 protein expression was reduced by TME yet unaffected by hypoxia, while Akt phosphorylation was increased by TME. The Akt loss was partly reversed by MCF-7 human breast cancer cell conditioned medium, suggesting that in vivo, the cancer cell secretome may compensate for adverse effects of TME on endothelial cells. TME, yet not hypoxia, reduced p70S6 kinase activity and ribosomal protein S6 phosphorylation and increased eIF2α phosphorylation, consistent with inhibition of protein translation. Finally, TME reduced Retinoblastoma protein phosphorylation and induced poly-ADP-ribose polymerase (PARP) cleavage consistent with inhibition of proliferation and induction of apoptosis. NHE1 knockdown, mimicking the effect of TME on NHE1 expression, reduced Ea.hy926 migration. TME effects on HIF-1α, VEGF, Akt, translation, proliferation or apoptosis markers were unaffected by NHE1 knockdown/inhibition. NHE1 and Akt are downregulated by TME conditions, more potently than by hypoxia alone. This inhibits endothelial cell migration and growth in a manner likely modulated by the cancer cell secretome.

Turbidity as a measure of salivary protein reactions with astringent substances.

PubMed

Horne, John; Hayes, John; Lawless, Harry T

2002-09-01

Binding of tannins to proline-rich proteins has been proposed as an initial step in the development of astringent sensations. In beer and fruit juices, formation of tannin-protein complexes leads to the well-known effect of haze development or turbidity. Two experiments examined the development of turbidity in human saliva when mixed with tannins as a potential in vitro correlate of astringent sensations. In the first study, haze was measured in filtered human saliva mixed with a range of tannic acid concentrations known to produce supra-threshold psychophysical responses. The second study examined relationships among individual differences in haze development and the magnitude of astringency ratings. Mostly negative correlations were found, consistent with the notion that high levels of salivary proteins protect oral tissues from the drying effects of tannic acid.

ADF Proteins Are Involved in the Control of Flowering and Regulate F-Actin Organization, Cell Expansion, and Organ Growth in Arabidopsis

PubMed Central

Dong, Chun-Hai; Xia, Gui-Xian; Hong, Yan; Ramachandran, Srinivasan; Kost, Benedikt; Chua, Nam-Hai

2001-01-01

Based mostly on the results of in vitro experiments, ADF (actin-depolymerizing factor) proteins are thought to be key modulators of the dynamic organization of the actin cytoskeleton. The few studies concerned with the in vivo function of ADF proteins that have been reported to date were performed almost exclusively using single-cell systems and have failed to produce consistent results. To investigate ADF functions in vivo and during the development of multicellular organs, we generated transgenic Arabidopsis plants that express a cDNA encoding an ADF protein (AtADF1) in the sense or the antisense orientation under the control of a strong constitutively active promoter. Selected lines with significantly altered levels of AtADF protein expression were characterized phenotypically. Overexpression of AtADF1 resulted in the disappearance of thick actin cables in different cell types, caused irregular cellular and tissue morphogenesis, and reduced the growth of cells and organs. In contrast, reduced AtADF expression promoted the formation of actin cables, resulted in a delay in flowering, and stimulated cell expansion as well as organ growth. These results are consistent with the molecular functions of ADF as predicted by in vitro studies, support the global roles of ADF proteins during the development of a multicellular organism, and demonstrate that these proteins are key regulators of F-actin organization, flowering, and cell and organ expansion in Arabidopsis. PMID:11402164

Effect of increasing energy and protein intake on body growth and carcass composition of heifer calves.

PubMed

Brown, E G; Vandehaar, M J; Daniels, K M; Liesman, J S; Chapin, L T; Keisler, D H; Nielsen, M S Weber

2005-02-01

The objective was to determine whether increased energy and protein intake between 2 and 14 wk of age would increase growth rates of heifer calves without fattening. At 2 wk of age, Holstein heifer calves were assigned to 1 of 4 treatments in a 2 x 2 factorial arrangement with 2 levels of protein and energy intake (moderate [M]; high [H]) in period 1 (2 to 8 wk of age) by 2 levels of protein and energy intake (low [L]; high [H]) in period 2 (8 to 14 wk of age) to produce similar initial BW for all 4 treatments. Treatments were ML, MH, HL, and HH, indicating moderate or high energy and protein intake during the first period and low or high intake during the second period. The M diet consisted of a standard milk replacer (21.3% CP, 21.3% fat) fed at 1.1% of BW on a DM basis and a 16.5% CP grain mix fed at restricted intake to promote 400 g of average daily gain (ADG), whereas the L diet consisted only of the grain mix. The H diet consisted of a high-protein milk replacer (30.3% CP, 15.9% fat) fed at 2% of BW on a DM basis and a 21.3% CP grain mix available ad libitum. Calves were weaned gradually from milk replacer by 7 wk and slaughtered at 8 (n = 11) or 14 wk of age (n = 41). In periods 1 and 2, ADG and the gain:feed ratio were greater for calves fed the H diet. Calves fed the H diet were taller after both periods 1 and 2. No difference was observed in carcass composition at 8 wk, but at 14 wk calves fed MH and HH had less water and more fat than calves fed ML and HL. Plasma IGF-I concentrations were greatest for calves fed the H diet during either period. Plasma leptin concentrations were increased in calves fed the H diet during period 1 from 4 to 6 wk of age. Increasing energy and protein intake from 2 to 8 wk and 8 to 14 wk of age increased BW, withers height, and gain:feed ratio. Calves fed the H diet from 8 to 14 wk of age had more body fat than calves fed the L diet. Increased energy and protein intake can increase the rate of body growth of heifer calves and potentially reduce rearing costs.

The malleable gut microbiome of juvenile rainbow trout (Oncorhynchus mykiss): Diet-dependent shifts of bacterial community structures

PubMed Central

Michl, Stéphanie Céline; Ratten, Jenni-Marie; Beyer, Matt; Hasler, Mario; LaRoche, Julie; Schulz, Carsten

2017-01-01

Plant-derived protein sources are the most relevant substitutes for fishmeal in aquafeeds. Nevertheless, the effects of plant based diets on the intestinal microbiome especially of juvenile Rainbow trout (Oncorhynchus mykiss) are yet to be fully investigated. The present study demonstrates, based on 16S rDNA bacterial community profiling, that the intestinal microbiome of juvenile Rainbow trout is strongly affected by dietary plant protein inclusion levels. After first feeding of juveniles with either 0%, 50% or 97% of total dietary protein content derived from plants, statistically significant differences of the bacterial gut community for the three diet-types were detected, both at phylum and order level. The microbiome of juvenile fish consisted mainly of the phyla Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria and Actinobacteria, and thus fits the salmonid core microbiome suggested in previous studies. Dietary plant proteins significantly enhanced the relative abundance of the orders Lactobacillales, Bacillales and Pseudomonadales. Animal proteins in contrast significantly promoted Bacteroidales, Clostridiales, Vibrionales, Fusobacteriales and Alteromonadales. The overall alpha diversity significantly decreased with increasing plant protein inclusion levels and with age of experimental animals. In order to investigate permanent effects of the first feeding diet-type on the early development of the microbiome, a diet change was included in the study after 54 days, but no such effects could be detected. Instead, the microbiome of juvenile trout fry was highly dependent on the actual diet fed at the time of sampling. PMID:28498878

The malleable gut microbiome of juvenile rainbow trout (Oncorhynchus mykiss): Diet-dependent shifts of bacterial community structures.

PubMed

Michl, Stéphanie Céline; Ratten, Jenni-Marie; Beyer, Matt; Hasler, Mario; LaRoche, Julie; Schulz, Carsten

2017-01-01

Plant-derived protein sources are the most relevant substitutes for fishmeal in aquafeeds. Nevertheless, the effects of plant based diets on the intestinal microbiome especially of juvenile Rainbow trout (Oncorhynchus mykiss) are yet to be fully investigated. The present study demonstrates, based on 16S rDNA bacterial community profiling, that the intestinal microbiome of juvenile Rainbow trout is strongly affected by dietary plant protein inclusion levels. After first feeding of juveniles with either 0%, 50% or 97% of total dietary protein content derived from plants, statistically significant differences of the bacterial gut community for the three diet-types were detected, both at phylum and order level. The microbiome of juvenile fish consisted mainly of the phyla Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria and Actinobacteria, and thus fits the salmonid core microbiome suggested in previous studies. Dietary plant proteins significantly enhanced the relative abundance of the orders Lactobacillales, Bacillales and Pseudomonadales. Animal proteins in contrast significantly promoted Bacteroidales, Clostridiales, Vibrionales, Fusobacteriales and Alteromonadales. The overall alpha diversity significantly decreased with increasing plant protein inclusion levels and with age of experimental animals. In order to investigate permanent effects of the first feeding diet-type on the early development of the microbiome, a diet change was included in the study after 54 days, but no such effects could be detected. Instead, the microbiome of juvenile trout fry was highly dependent on the actual diet fed at the time of sampling.

Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

NASA Astrophysics Data System (ADS)

Luo, Zhang; Liu, Zhixin; Fu, Jianping; Zhang, Qiusheng; Huang, Bei; Nie, Pin

2016-11-01

Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes ( IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon ( IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.

Effects of replacing rapeseed meal with fava bean at 2 concentrate crude protein levels on feed intake, nutrient digestion, and milk production in cows fed grass silage-based diets.

PubMed

Puhakka, L; Jaakkola, S; Simpura, I; Kokkonen, T; Vanhatalo, A

2016-10-01

The objective of this study was to evaluate the production and physiological responses of dairy cows to the substitution of fava bean for rapeseed meal at 2 protein supplementation levels in grass silage-based diets. We used 6 primiparous and 6 multiparous Finnish Ayrshire cows in a cyclic changeover trial with a 2×3 factorial arrangement of treatments. The experimental diets consisted of formic acid-treated timothy-meadow fescue silage and 3 isonitrogenous concentrates containing either rapeseed meal, fava bean, or a 1:1 mixture of rapeseed meal and fava bean at low and high inclusion rates, resulting in concentrate crude protein (CP) levels of 15.4 and 19.0% in dry matter. Silage dry matter intake decreased linearly when rapeseed meal was replaced with fava bean, the negative effect being more distinct at the high CP level than the low (-2.3 vs. -0.9kg/d, respectively). Similarly, milk and milk protein yields decreased linearly with fava bean, the change tending to be greater at the high CP level than the low. Yield of milk fat was lower for fava bean compared with rapeseed meal, the difference showing no interaction with CP level. Especially at the high CP level, milk urea concentration was higher with fava bean compared with rapeseed meal indicating better utilization of protein from the rapeseed meal. The apparent total-tract organic matter digestibility did not differ between treatments at the low CP level, but digestibility was higher for fava bean than for rapeseed meal at the high CP level. Plasma concentrations of essential amino acids, including methionine and lysine, were lower for fava bean than for rapeseed meal. Compared with rapeseed meal, the use of fava bean in dairy cow diets as the sole protein supplement decreased silage intake and milk production in highly digestible formic acid-treated grass silage-based diets. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

[EFFECT OF EARLY PROTEIN-CALORIE MALNUTRITION ON NUTRITIONAL STATUS AND ATTRIBUTES OF THE METABOLIC SYNDROME IN YOUNG ADULTS].

PubMed

Escaffi Fonseca, María José; Moreira Carrasco, Loreto; Rodríguez Osiac, Lorena; Pizarro Quevedo, Tito; Cavada Chacón, Gabriel; Villarroel del Pino, Luis; Salas Guzmán, Natalia; Muzzo Benavides, Santiago; Mönckeberg Barros, Fernando; Rozowski Narkunska, Jaime; Castillo Valenzuela, Oscar

2015-09-01

during recent years consistent studies have characterized the relationship between moderate and severe protein-calorie malnutrition and the appearance of non-communicable diseases in adulthood like metabolic syndrome (MS). to analyze the relationship between moderate and severe protein-calorie malnutrition during the first 1 000 days of life and the MS in a cohort of adults from Curicó, Chile. we studied 49 young adults who had suffered moderate to severe protein-calorie malnutrition during their first two years of life. Anthropometry, blood pressure measurement and laboratory tests were performed, and the burden of MS attributes was determined. the prevalence of MS was 14.3% with no significant differences by gender, showing a direct and significant association between burden of MS and body mass index, waist / height index, blood pressure, plasma levels of glucose and triglyceride, and an inverse association with HDL. systolic blood pressure and plasma level of triglyceride represented the most important risk factors for SM in this cohort. We found no association between the presence of protein-calorie malnutrition and MS. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

Physiologically Based Pharmacokinetic Modeling of Therapeutic Proteins.

PubMed

Wong, Harvey; Chow, Timothy W

2017-09-01

Biologics or therapeutic proteins are becoming increasingly important as treatments for disease. The most common class of biologics are monoclonal antibodies (mAbs). Recently, there has been an increase in the use of physiologically based pharmacokinetic (PBPK) modeling in the pharmaceutical industry in drug development. We review PBPK models for therapeutic proteins with an emphasis on mAbs. Due to their size and similarity to endogenous antibodies, there are distinct differences between PBPK models for small molecules and mAbs. The high-level organization of a typical mAb PBPK model consists of a whole-body PBPK model with organ compartments interconnected by both blood and lymph flows. The whole-body PBPK model is coupled with tissue-level submodels used to describe key mechanisms governing mAb disposition including tissue efflux via the lymphatic system, elimination by catabolism, protection from catabolism binding to the neonatal Fc (FcRn) receptor, and nonlinear binding to specific pharmacological targets of interest. The use of PBPK modeling in the development of therapeutic proteins is still in its infancy. Further application of PBPK modeling for therapeutic proteins will help to define its developing role in drug discovery and development. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Improving protein-protein interaction prediction using evolutionary information from low-quality MSAs.

PubMed

Várnai, Csilla; Burkoff, Nikolas S; Wild, David L

2017-01-01

Evolutionary information stored in multiple sequence alignments (MSAs) has been used to identify the interaction interface of protein complexes, by measuring either co-conservation or co-mutation of amino acid residues across the interface. Recently, maximum entropy related correlated mutation measures (CMMs) such as direct information, decoupling direct from indirect interactions, have been developed to identify residue pairs interacting across the protein complex interface. These studies have focussed on carefully selected protein complexes with large, good-quality MSAs. In this work, we study protein complexes with a more typical MSA consisting of fewer than 400 sequences, using a set of 79 intramolecular protein complexes. Using a maximum entropy based CMM at the residue level, we develop an interface level CMM score to be used in re-ranking docking decoys. We demonstrate that our interface level CMM score compares favourably to the complementarity trace score, an evolutionary information-based score measuring co-conservation, when combined with the number of interface residues, a knowledge-based potential and the variability score of individual amino acid sites. We also demonstrate, that, since co-mutation and co-complementarity in the MSA contain orthogonal information, the best prediction performance using evolutionary information can be achieved by combining the co-mutation information of the CMM with co-conservation information of a complementarity trace score, predicting a near-native structure as the top prediction for 41% of the dataset. The method presented is not restricted to small MSAs, and will likely improve interface prediction also for complexes with large and good-quality MSAs.

Identification of marker proteins for the adulteration of meat products with soybean proteins by multidimensional liquid chromatography-tandem mass spectrometry.

PubMed

Leitner, Alexander; Castro-Rubio, Florentina; Marina, Maria Luisa; Lindner, Wolfgang

2006-09-01

Soybean proteins are frequently added to processed meat products for economic reasons and to improve their functional properties. Monitoring of the addition of soybean protein to meat products is of high interest due to the existence of regulations forbidding or limiting the amount of soybean proteins that can be added during the processing of meat products. We have used chromatographic prefractionation on the protein level by perfusion liquid chromatography to isolate peaks of interest from extracts of soybean protein isolate (SPI) and of meat products containing SPI. After enzymatic digestion using trypsin, the collected fractions were analyzed by nanoflow liquid chromatography-tandem mass spectrometry. Several variants and subunits of the major seed proteins, glycinin and beta-conglycinin, were identified in SPI, along with two other proteins. In soybean-protein-containing meat samples, different glycinin A subunits could be identified from the peak discriminating between samples with and without soybean proteins added. Among those, glycinin G4 subunit A4 was consistently found in all samples. Consequently, this protein (subunit) can be used as a target for new analytical techniques in the course of identifying the addition of soybean protein to meat products.

The Cancer-Related Transcription Factor Runx2 Modulates Cell Proliferation in Human Osteosarcoma Cell Lines

PubMed Central

Lucero, Claudia M.J.; Vega, Oscar A.; Osorio, Mariana M.; Tapia, Julio C.; Antonelli, Marcelo; Stein, Gary S.; Van Wijnen, Andre J.; Galindo, Mario A.

2013-01-01

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G1/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G1/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. PMID:22949168

Kinetics of nif Gene Expression in a Nitrogen-Fixing Bacterium

PubMed Central

Poza-Carrión, César; Jiménez-Vicente, Emilio; Navarro-Rodríguez, Mónica; Echavarri-Erasun, Carlos

2014-01-01

Nitrogen fixation is a tightly regulated trait. Switching from N2 fixation-repressing conditions to the N2-fixing state is carefully controlled in diazotrophic bacteria mainly because of the high energy demand that it imposes. By using quantitative real-time PCR and quantitative immunoblotting, we show here how nitrogen fixation (nif) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA, was followed by subsequent, longer-duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, expression levels, and NifA dependence varied greatly among the nif operons. Moreover, the exact concentrations of Nif proteins and their changes over time were determined for the first time. Nif protein concentrations were exquisitely balanced, with FeMo cofactor biosynthetic proteins accumulating at levels 50- to 100-fold lower than those of the structural proteins. Mutants lacking nitrogenase structural genes or impaired in FeMo cofactor biosynthesis showed overenhanced responses to derepression that were proportional to the degree of nitrogenase activity impairment, consistent with the existence of at least two negative-feedback regulatory mechanisms. The first such mechanism responded to the levels of fixed nitrogen, whereas the second mechanism appeared to respond to the levels of the mature NifDK component. Altogether, these findings provide a framework to engineer N2 fixation in nondiazotrophs. PMID:24244007

Kinetics of Nif gene expression in a nitrogen-fixing bacterium.

PubMed

Poza-Carrión, César; Jiménez-Vicente, Emilio; Navarro-Rodríguez, Mónica; Echavarri-Erasun, Carlos; Rubio, Luis M

2014-02-01

Nitrogen fixation is a tightly regulated trait. Switching from N2 fixation-repressing conditions to the N2-fixing state is carefully controlled in diazotrophic bacteria mainly because of the high energy demand that it imposes. By using quantitative real-time PCR and quantitative immunoblotting, we show here how nitrogen fixation (nif) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA, was followed by subsequent, longer-duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, expression levels, and NifA dependence varied greatly among the nif operons. Moreover, the exact concentrations of Nif proteins and their changes over time were determined for the first time. Nif protein concentrations were exquisitely balanced, with FeMo cofactor biosynthetic proteins accumulating at levels 50- to 100-fold lower than those of the structural proteins. Mutants lacking nitrogenase structural genes or impaired in FeMo cofactor biosynthesis showed overenhanced responses to derepression that were proportional to the degree of nitrogenase activity impairment, consistent with the existence of at least two negative-feedback regulatory mechanisms. The first such mechanism responded to the levels of fixed nitrogen, whereas the second mechanism appeared to respond to the levels of the mature NifDK component. Altogether, these findings provide a framework to engineer N2 fixation in nondiazotrophs.

TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats.

PubMed

Sun, Xiaoyan; Song, Ming; Wang, Hui; Zhou, Huimin; Wang, Feng; Li, Ya; Zhang, Yun; Zhang, Wei; Zhong, Ming; Ti, Yun

2017-06-17

Our previous study had suggested Tribbles homolog 3 (TRB3) might be involved in metabolic syndrome via adipose tissue. Given prior studies, we sought to determine whether TRB3 plays a major role in adipocytes and adipose tissue with beneficial metabolic effects in obese and diabetic rats. Fully differentiated 3T3-L1 adipocytes were incubated to induce insulin resistant adipocytes. Forty male Sprague-Dawley rats were all fed high-fat (HF) diet. Type 2 diabetic rat model was induced by high-fat diet and low-dose streptozotocin (STZ). Compared with control group, in insulin resistant adipocytes, protein levels of insulin receptor substrate-1(IRS-1), glucose transporter 4(GLUT4) and phosphorylated-AMP-activated protein kinase (p-AMPK)were reduced, TRB3 protein level and triglyceride level were significantly increased, glucose uptake was markedly decreased. TRB3 silencing alleviated adipocytes insulin resistance. With TRB3 gene silencing, protein levels of IRS-1, GLUT4 and p-AMPK were significantly increased in adipocytes. TRB3 gene silencing decreased blood glucose, ameliorated insulin sensitivity and adipose tissue remodeling in diabetic rats. TRB3 silencing decreased triglyceride, increased glycogen simultaneously in diabetic epididymal and brown adipose tissues (BAT). Consistently, p-AMPK levels were increased in diabetic epididymal adipose tissue, and BAT after TRB3-siRNA treatment. TRB3silencing increased phosphorylation of Akt in liver, and improved liver insulin resistance. Copyright © 2017. Published by Elsevier Inc.

Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry.

PubMed

Hou, Yong; Zhang, Yan; Gong, Jing; Tian, Sha; Li, Jianwei; Dong, Zhaoming; Guo, Chao; Peng, Li; Zhao, Ping; Xia, Qingyou

2016-05-01

The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used LC-MS/MS to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the second day of wandering stage, first day of pupation, ninth day of pupation, and first day as an adult moth. GO annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity-related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant-binding, and chemosensory proteins, showed stage-specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

G protein-coupled receptor kinase 2 positively regulates epithelial cell migration

PubMed Central

Penela, Petronila; Ribas, Catalina; Aymerich, Ivette; Eijkelkamp, Niels; Barreiro, Olga; Heijnen, Cobi J; Kavelaars, Annemieke; Sánchez-Madrid, Francisco; Mayor, Federico

2008-01-01

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration. PMID:18369319

Identification of Genes Encoding Granule-Bound Starch Synthase Involved in Amylose Metabolism in Banana Fruit

PubMed Central

Liu, Weixin; Xu, Biyu; Jin, Zhiqiang

2014-01-01

Granule-bound starch synthase (GBSS) is responsible for amylose synthesis, but the role of GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage. PMID:24505384

Heat shock protein defenses in the neo- and allocortex of the telencephalon

PubMed Central

Posimo, Jessica M.; Weilnau, Justin N.; Gleixner, Amanda M.; Broeren, Matthew T.; Weiland, Nicole L.; Brodsky, Jeffrey L.; Wipf, Peter; Leak, Rehana K.

2015-01-01

The telencephalic allocortex develops protein inclusions before the neocortex in many age-related proteinopathies. One major defense mechanism against proteinopathic stress is the heat shock protein (Hsp) network. We therefore contrasted Hsp defenses in stressed primary neo- and allocortical cells. Neocortical neurons were more resistant to the proteasome inhibitor MG132 than neurons from three allocortical subregions: entorhinal cortex, piriform cortex, and hippocampus. However, allocortical neurons exhibited higher MG132-induced increases in Hsp70 and Hsc70. MG132-treated allocortical neurons also exhibited greater levels of protein ubiquitination. Inhibition of Hsp70/Hsc70 activity synergistically exacerbated MG132 toxicity in allocortical neurons more than neocortical neurons, suggesting that the allocortex is more reliant on these Hsp defenses. In contrast, astrocytes harvested from neo- or allocortex did not differ in their response to Hsp70/Hsc70 inhibition. Consistent with the idea that chaperones are maximally engaged in allocortical neurons, an increase in Hsp70/Hsc70 activity was protective only in neocortical neurons. Finally, the levels of select Hsps were altered in neocortex and allocortex in vivo with aging. PMID:25771395

Quantum studies of deprotonated forms of malonic acid

NASA Astrophysics Data System (ADS)

Asciutto, Eliana; Lee, Jung Goo; Pedersen, Lee G.; Sagui, Celeste

2004-03-01

There have been numerous computational studies on carboxylic acids, specially in the simplest ones (formic and acetic acids). A considerable amount of these computations has been dedicated towards developing an understanding of proton transfer. In this work we study malonate as a model for γ-carboxyglutamic acid (Gla). Gla is a metal-binding amino acid whose synthesis is dependent upon vitamin K. Of the classes of proteins that contain Gla, the vitamin K-dependent blood coagulation and regulatory proteins have have been most thoroughly studied. The Gla domain in these proteins (generally consisting of 10-12 Gla residues) induces an structure that facilitates calcium-mediated interactions of the protein with membrane surfaces. In order to get a better understanding of this fundamental role of Gla at a quantum level, we study the role of intramolecular proton transfer in malonate in its divalent anionic form. The di-anion is particularly challenging. A correct description of the potential energy hypersurface is obtained only by application of large basis sets in conjunction with methods including high-level treatment of electron correlation effects.

Cysteic Acid in Dietary Keratin is Metabolized to Glutathione and Liver Taurine in a Rat Model of Human Digestion

PubMed Central

Wolber, Frances M.; McGrath, Michelle; Jackson, Felicity; Wylie, Kim; Broomfield, Anne

2016-01-01

Poultry feathers, consisting largely of keratin, are a low-value product of the poultry industry. The safety and digestibility of a dietary protein produced from keratin (KER) was compared to a cysteine-supplemented casein-based diet in a growing rat model for four weeks. KER proved to be an effective substitute for casein at 50% of the total dietary protein, with no changes in the rats’ food intake, weight gain, organ weight, bone mineral density, white blood cell counts, liver glutathione, or blood glutathione. Inclusion of KER in the diet reduced total protein digestibility from 94% to 86% but significantly increased total dietary cysteine uptake and subsequent liver taurine levels. The KER diet also significantly increased caecum weight and significantly decreased fat digestibility, resulting in a lower proportion of body fat, and induced a significant increase in blood haemoglobin. KER is therefore a safe and suitable protein substitute for casein, and the cysteic acid in keratin is metabolised to maintain normal liver and blood glutathione levels. PMID:26907334

Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size.

PubMed

Anema, Skelte G; Li, Yuming

2003-02-01

When skim milk at pH 6.55 was heated (75 to 100 degrees C for up to 60 min), the casein micelle size, as monitored by photon correlation spectroscopy, was found to increase during the initial stages of heating and tended to plateau on prolonged heating. At any particular temperature, the casein micelle size increased with longer holding times, and, at any particular holding time, the casein micelle size increased with increasing temperature. The maximum increase in casein micelle size was about 30-35 nm. The changes in casein micelle size were poorly correlated with the level of whey protein denaturation. However, the changes in casein micelle size were highly correlated with the levels of denatured whey proteins that were associated with the casein micelles. The rate of association of the denatured whey proteins with the casein micelles was considerably slower than the rate of denaturation of the whey proteins. Removal of the whey proteins from the skim milk resulted in only small changes in casein micelle size during heating. Re-addition of beta-lactoglobulin to the whey-protein-depleted milk caused the casein micelle size to increase markedly on heat treatment. The changes in casein micelle size induced by the heat treatment of skim milk may be a consequence of the whey proteins associating with the casein micelles. However, these associated whey proteins would need to occlude a large amount of serum to account for the particle size changes. Separate experiments showed that the viscosity changes of heated milk and the estimated volume fraction changes were consistent with the particle size changes observed. Further studies are needed to determine whether the changes in size are due to the specific association of whey proteins with the micelles or whether a low level of aggregation of the casein micelles accompanies this association behaviour. Preliminary studies indicated lower levels of denatured whey proteins associated with the casein micelles and smaller changes in casein micelle size occurred as the pH of the milk was increased from pH 6.5 to pH 6.7.

Maternal fructose intake disturbs ovarian estradiol synthesis in rats.

PubMed

Munetsuna, Eiji; Yamada, Hiroya; Yamazaki, Mirai; Ando, Yoshitaka; Mizuno, Genki; Ota, Takeru; Hattori, Yuji; Sadamoto, Nao; Suzuki, Koji; Ishikawa, Hiroaki; Hashimoto, Shuji; Ohashi, Koji

2018-06-01

Recent increases in fructose consumption have raised concerns regarding the potential adverse intergenerational effects, as maternal fructose intake may induce physiological dysfunction in offspring. However, no reports are available regarding the effect of excess maternal fructose on reproductive tissues such as the ovary. Notably, the maternal intrauterine environment has been demonstrated to affect ovarian development in the subsequent generation. Given the fructose is transferred to the fetus, excess fructose consumption may affect offspring ovarian development. As ovarian development and its function is maintained by 17β-estradiol, we therefore investigated whether excess maternal fructose intake influences offspring ovarian estradiol synthesis. Rats received a 20% fructose solution during gestation and lactation. After weaning, offspring ovaries were isolated. Offspring from fructose-fed dams showed reduced StAR and P450(17α) mRNA levels, along with decreased protein expression levels. Conversely, attenuated P450arom protein level was found in the absence of mRNA expression alteration. Consistent with these phenomena, decreased circulating levels of estradiol were observed. Furthermore, estrogen receptor α (ERα) protein levels were also down-regulated. In accordance, the mRNA for progesterone receptor, a transcriptional target of ERα, was decreased. These results suggest that maternal fructose might alter ovarian physiology in the subsequent generation. Copyright © 2018 Elsevier Inc. All rights reserved.

Heart type fatty acid binding protein response and subsequent development of atherosclerosis in insulin resistant polycystic ovary syndrome patients.

PubMed

Cakir, Evrim; Ozbek, Mustafa; Sahin, Mustafa; Cakal, Erman; Gungunes, Askin; Ginis, Zeynep; Demirci, Taner; Delibasi, Tuncay

2012-12-18

Women with polycystic ovary syndrome (PCOS) have higher risk for cardiovascular disease (CVD). Heart type fatty acid binding protein (HFABP) has been found to be predictive for myocardial ischemia.Wet ested whether HFABP is the predictor for CVD in PCOS patients, who have an increased risk of cardiovascular disease. This was a prospective, cross sectional controlled study conducted in a training and research hospital.The study population consisted of 46 reproductive-age PCOS women and 28 control subjects. We evaluated anthropometric and metabolic parameters, carotid intima media thickness and HFABP levels in both PCOS patients and control group. Mean fasting insulin, homeostasis model assessment insulin resistance index (HOMA-IR), triglyceride, total cholesterol, low density lipoprotein cholesterol, free testosterone, total testosterone, carotid intima media thickness (CIMT) levels were significantly higher in PCOS patients. Although HFABP levels were higher in PCOS patients, the difference did not reach statistically significant in early age groups. After adjustment for age and body mass index, HFABP level was positive correlated with hsCRP, free testosterone levels, CIMT and HOMA-IR. Heart type free fatty acid binding protein appeared to have an important role in metabolic response and subsequent development of atherosclerosis in insulin resistant, hyperandrogenemic PCOS patients.

Choline acetyltransferase expression during periods of behavioral activity and across natural sleep-wake states in the basal forebrain.

PubMed

Greco, M A; McCarley, R W; Shiromani, P J

1999-01-01

The present study examined whether the expression of the messenger RNA encoding the protein responsible for acetylcholine synthesis is associated with sleep-wakefulness. Choline acetyltransferase messenger RNA levels were analysed using a semi-quantitative assay in which reverse transcription was coupled to complementary DNA amplification using the polymerase chain reaction. To examine the relationship between steady-state messenger RNA and behavioral activity, rats were killed during the day (4.00 p.m.) or night (4.00 a.m.), and tissue from the vertical and horizontal limbs of the diagonal bands of Broca was analysed. Choline acetyltransferase messenger RNA levels were higher during the day than during the night. The second study examined more closely the association between choline acetyltransferase messenger RNA levels and individual bouts of wakefulness, slow-wave sleep or rapid eye movement sleep. Choline acetyltransferase messenger RNA levels were low during wakefulness, intermediate in slow-wave sleep and high during rapid eye movement sleep. In contrast, protein activity, measured at a projection site of cholinergic neurons of the basal forebrain, was higher during wakefulness than during sleep. These findings suggest that choline acetyltransferase protein and messenger RNA levels exhibit an inverse relationship during sleep and wakefulness. The increased messenger RNA expression during sleep is consistent with a restorative function of sleep.

High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

PubMed

Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

2016-03-01

Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (~7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A comparative secretome analysis of industrial Aspergillus oryzae and its spontaneous mutant ZJGS-LZ-21.

PubMed

Zhu, Yuanyuan; Liang, Xinle; Zhang, Hong; Feng, Wei; Liu, Ye; Zhang, Fuming; Linhardt, Robert J

2017-05-02

Aspergillus oryzae koji plays a crucial role in fermented food products due to the hydrolytic activities of secreted enzymes. In the present study, we performed a comparative secretome analysis of the industrial strain of Aspergillus oryzae 3.042 and its spontaneous mutantZJGS-LZ-21. One hundred and fifty two (152) differential protein spots were excised (p<0.05), and 25 proteins were identified. Of the identified proteins, 91.3% belonged to hydrolytic enzymes acting on carbohydrates or proteins. Consistent with their enzyme activities, the expression of 14 proteins involved in the degradation of cellulose, hemicellulose, starch and proteins, increased in the ZJGS-LZ-21isolate. In particular, increased levels of acid protease (Pep) may favor the degradation of soy proteins in acidic environments and promote the cleavage of allergenic soybean proteins in fermentation, resulting in improvements of product safety and quality. The ZJGS-LZ-21 isolate showed higher protein secretion and increased hydrolytic activities than did strain 3.042, indicating its promising application in soybean paste fermentation. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of dietary protein and GABA on food intake, growth and tissue amino acids in cats.

PubMed

Tews, J K; Rogers, Q R; Morris, J G; Harper, A E

1984-02-01

GABA at 5%, but not 3%, of a low protein diet depressed food intake and growth of kittens. Adaptation to high protein prevented these effects. When cats adapted to low or high protein were fed a meal containing GABA, plasma GABA concentration after 2 hr was 8-fold higher in the low than in the high protein group; clearance was almost complete within 6 hr. Concentrations of proline, branched-chain, other large neutral and basic (especially ornithine) amino acids increased more when cats were fed a high rather than a low protein meal; glycine decreased. At 6 hr, concentrations had consistently returned to initial levels only in the low protein group. Feeding the high protein diet ad lib increased tissue concentrations of threonine, proline and the branched-chain amino acids. Hepatic or renal GABA-aminotransferase activity was not altered in kittens fed the high protein diet. Kidney activity was 10-fold that of liver, which may contribute to the better tolerance of GABA by cats than by rats.

Serum C-reactive protein in the prediction of cardiovascular diseases: Overview of the latest clinical studies and public health practice.

PubMed

Avan, Amir; Tavakoly Sany, Seyedeh Belin; Ghayour-Mobarhan, Majid; Rahimi, Hamid Reza; Tajfard, Mohammad; Ferns, Gordon

2018-06-22

Cardiovascular disease is the most common cause of morbidity and mortality globally. Epidemiological studies using high-sensitivity assays for serum C-reactive protein have shown a consistent association between cardiovascular disease risk and serum C-reactive protein concentrations. C-reactive protein is a biomarker for inflammation, and has been established in clinical practice as an independent risk factor for cardiovascular disease events. There is evidence that serum C-reactive protein is an excellent biomarker of cardiovascular disease and is also an independent and strong predictor of adverse cardiovascular events. Further characterization of the impact and influence of lifestyle exposures and genetic variation on the C-reactive protein response to cardiovascular disease events may have implications for the therapeutic approaches to reduce cardiovascular disease events. This review summarizes the studies that have examined the association between serum C-reactive protein and the risk of cardiovascular disease. We also discuss the impact of independent factors and C-reactive protein genetic polymorphisms on baseline plasma C-reactive protein levels. © 2018 Wiley Periodicals, Inc.

Methylmercury alters glutathione homeostasis by inhibiting glutaredoxin 1 and enhancing glutathione biosynthesis in cultured human astrocytoma cells.

PubMed

Robitaille, Stephan; Mailloux, Ryan J; Chan, Hing Man

2016-08-10

Methylmercury (MeHg) is a neurotoxin that binds strongly to thiol residues on protein and low molecular weight molecules like reduced glutathione (GSH). The mechanism of its effects on GSH homeostasis particularly at environmentally relevant low doses is not fully known. We hypothesized that exposure to MeHg would lead to a depletion of reduced glutathione (GSH) and an accumulation of glutathione disulfide (GSSG) leading to alterations in S-glutathionylation of proteins. Our results showed exposure to low concentrations of MeHg (1μM) did not significantly alter GSH levels but increased GSSG levels by ∼12-fold. This effect was associated with a significant increase in total cellular glutathione content and a decrease in GSH/GSSG. Immunoblot analyses revealed that proteins involved in glutathione synthesis were upregulated accounting for the increase in cellular glutathione. This was associated an increase in cellular Nrf2 protein levels which is required to induce the expression of antioxidant genes in response to cellular stress. Intriguingly, we noted that a key enzyme involved in reversing protein S-glutathionylation and maintaining glutathione homeostasis, glutaredoxin-1 (Grx1), was inhibited by ∼50%. MeHg treatment also increased the S-glutathionylation of a high molecular weight protein. This observation is consistent with the inhibition of Grx1 and elevated H2O2 production however; contrary to our original hypothesis we found few S-glutathionylated proteins in the astrocytoma cells. Collectively, MeHg affects multiple arms of glutathione homeostasis ranging from pool management to protein S-glutathionylation and Grx1 activity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Expression of the B subunit of Escherichia coli heat-labile enterotoxin as a fusion protein in transgenic tomato.

PubMed

Walmsley, A M; Alvarez, M L; Jin, Y; Kirk, D D; Lee, S M; Pinkhasov, J; Rigano, M M; Arntzen, C J; Mason, H S

2003-06-01

Epitopes often require co-delivery with an adjuvant or targeting protein to enable recognition by the immune system. This paper reports the ability of transgenic tomato plants to express a fusion protein consisting of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) and an immunocontraceptive epitope. The fusion protein was found to assemble into pentamers, as evidenced by its ability to bind to gangliosides, and had an average expression level of 37.8 microg g(-1) in freeze-dried transgenic tissues. Processing of selected transgenic fruit resulted in a 16-fold increase in concentration of the antigen with minimal loss in detectable antigen. The species-specific nature of this epitope was shown by the inability of antibodies raised against non-target species to detect the LTB fusion protein. The immunocontraceptive ability of this vaccine will be tested in future pilot mice studies.

Effect of addition of ripe bananas on some physico-chemical properties of maize 'extract'.

PubMed

Okezie, U; Akanbi, C T; Otunola, E T; Adeyemi, I A

2003-11-01

Flour mixes obtained by the addition of banana pulp in various proportions (0-50%) to maize 'extracts' were evaluated for some quality characteristics. All the mixes had significantly lower values of crude protein, fat and water-holding capacity. Gelation, however, significantly increased the water-holding capacity in all cases. The ash content, titratable acidity and total sugars increased tremendously with an increase in the level of banana substitution. While both Adam's consistency values and equilibrium moisture content decreased with an increase in the level of banana substitution, the syneresis values did not show any consistent pattern. The consistently low moisture content and the results of the moisture sorption isotherms suggest a good storage stability of all the mixes, especially when kept under conditions of water activity of 0.30 and below, and their possible suitability for baked products.

Effect of increasing energy and protein intake on mammary development in heifer calves.

PubMed

Brown, E G; Vandehaar, M J; Daniels, K M; Liesman, J S; Chapin, L T; Forrest, J W; Akers, R M; Pearson, R E; Nielsen, M S Weber

2005-02-01

The objective of this study was to determine if increased energy and protein intake from 2 to 14 wk of age would affect mammary development in heifer calves. At 2 wk of age, Holstein heifer calves were assigned to 1 of 4 treatments in a 2 x 2 factorial arrangement with 2 levels of protein and energy intake (moderate, M; high, H) in period 1 (2 to 8 wk of age) and 2 levels of protein and energy intake (low, L; high, H) in period 2 (8 to 14 wk of age), so that mean initial body weights were approximately equal for all 4 treatments (ML, MH, HL, and HH). The M diet in period 1 consisted of a standard milk replacer (21.3% CP, 21.3% fat) fed at 1.1% of BW on a DM basis and a 16.5% CP grain mix fed at restricted intake to promote 400 g of daily gain, whereas the L diet in period 2 consisted only of the grain mix. The H diet in period 1 consisted of a high-protein milk replacer (30.3% CP, 15.9% fat) fed at 2.0% of body weight on a DM basis and a 21.3% CP grain mix available ad libitum. In period 2, the H diet consisted of just the 21.3% grain mix. Calves were gradually weaned from milk replacer by 7 wk and slaughtered at 8 (n = 11) or 14 wk of age (n = 41). Parenchyma from the distal region, midgland, and proximal region relative to the teat from one half of the udder was collected, fixed, and embedded in paraffin. The other half of the gland was used to determine parenchymal mass, protein, fat, DNA, RNA, and extraparenchymal mass. Total parenchymal tissue, parenchymal DNA, parenchymal RNA, and concentrations of DNA and RNA were higher for calves on the H diet during period 1, but were not affected by diet during period 2. Parenchymal fat percentage was increased by the H diet during period 2. The H diet increased extraparenchymal fat during both periods. The area of parenchyma occupied by epithelium was not affected by treatment, but at the end of period 2, the percentage of proliferating epithelial cells as indicated by Ki67, an marker of cell proliferation, expression was greater for calves on the M diet in period 1 compared with calves on the H diet in period 1. Diets did not influence parenchymal protein percentage or the ratio of RNA to DNA. Higher energy and protein intake from 2 to 8 wk of age increased parenchymal mass and parenchymal DNA and RNA in mammary glands of heifer calves without increasing deposition of parenchymal fat. Diet also influenced histological development of mammary parenchyma and subsequent proliferation of ductal epithelial cells. Implications of these effects for future milk production potential are unknown.

Differences in osmotolerance in freshwater and brackish water populations of Theodoxus fluviatilis (Gastropoda: Neritidae) are associated with differential protein expression.

PubMed

Symanowski, Frauke; Hildebrandt, Jan-Peter

2010-03-01

The euryhaline gastropod Theodoxus fluviatilis is found in northern Germany in freshwater or in brackish water habitats in the Baltic Sea. Previous studies have revealed that individuals from both habitats are not distinguishable by morphological characters or by sequence comparison of DNA encoding 16S RNA or cytochrome C. As reported in this study, animals collected in the two habitats differ substantially in their physiological ability to adapt to different salinities. Comparison of accumulation rates of ninhydrin-positive substances (NPS) in foot muscle upon transfer of animals to higher medium salinities revealed that brackish water animals were perfectly able to mobilize NPS, while freshwater animals had only limited ability to do so. In an attempt to explore whether this difference in physiology may be caused by genetic differentiation, we compared protein expression patterns of soluble foot muscle proteins using 2D gel electrophoresis and silver staining. Of the 40 consistently detected protein spots, 27 showed similar levels in protein expression in animals collected from freshwater or brackish water habitats, respectively. In 12 spots, however, protein concentration was higher in brackish water than in freshwater animals. In four of these spots, expression levels followed increases or decreases in medium salinities. In a different set of 4 of these 12 spots, protein levels were always higher in brackish water as compared to freshwater animals, regardless of their physiological situation (14 days in artificial pond water or in medium with a salinity of 16 per thousand). The remaining 4 of the 12 spots had complex expression patterns. Protein levels of the remaining single spot were generally higher in freshwater animals than in brackish water animals. These expression patterns may indicate that freshwater and brackish water animals of T. fluviatilis belong to different locally adapted populations with subtle genetic differentiation.

Cloning of a long HIV-1 readthrough transcript and detection of an increased level of early growth response protein-1 (Egr-1) mRNA in chronically infected U937 cells.

PubMed

Dron, M; Hameau, L; Benboudjema, L; Guymarho, J; Cajean-Feroldi, C; Rizza, P; Godard, C; Jasmin, C; Tovey, M G; Lang, M C

1999-01-01

To identify the pathways involved in HIV-1 modification of cellular gene expression, chronically infected U937 cells were screened by mRNA differential display. A chimeric transcript consisting of the 3' end of the LTR of a HIV-1 provirus, followed by 3.7 kb of cellular RNA was identified suggesting that long readthrough transcription might be one of the mechanisms by which gene expression could be modified in individual infected cells. Such a phenomenon may also be the first step towards the potential transduction of cellular sequences. Furthermore, the mRNA encoding for the transcription factor Egr-1 was detected as an over-represented transcript in infected cells. Northern blot analysis confirmed the increase of Egr-1 mRNA content in both HIV-1 infected promonocytic U937 cells and T cell lines such as Jurkat and CEM. Interestingly a similar increase of Egr-1 mRNA has previously been reported to occur in HTLV-1 and HTLV-2 infected T cell lines. Despite the consistent increase in the level of Egr-1 mRNA, the amount of the encoded protein did not appear to be modified in HIV-1 infected cells, suggesting an increased turn over of the protein in chronically infected cells.

Levels of plasma ceruloplasmin protein are markedly lower following dietary copper deficiency in rodents

PubMed Central

Broderius, Margaret; Mostad, Elise; Wendroth, Krista; Prohaska, Joseph R.

2010-01-01

Ceruloplasmin (Cp) is a multicopper oxidase and the most abundant copper binding protein in vertebrate plasma. Loss of function mutations in humans or experimental deletion in mice result in iron overload consistent with a putative ferroxidase function. Prior work suggested plasma may contain multiple ferroxidases. Studies were conducted in Holtzman rats (Rattus novegicus), albino mice (Mus musculus), Cp -/- mice, and adult humans (Homo sapiens) to investigate the copper-iron interaction. Dietary copper-deficient (CuD) rats and mice were produced using a modified AIN-76A diet. Results confirmed that o-dianisidine is a better substrate than paraphenylene diamine (PPD) for assessing diamine oxidase activity of Cp. Plasma from CuD rat dams and pups, and CuD and Cp -/- mice contained no detectable Cp diamine oxidase activity. Importantly, no ferroxidase activity was detectable for CuD rats, mice, or Cp -/- mice compared to robust activity for copper-adequate (CuA) rodent controls using western membrane assay. Immunoblot protocols detected major reductions (60-90%) in Cp protein in plasma of CuD rodents but no alteration in liver mRNA levels by qRT-PCR. Data are consistent with apo-Cp being less stable than holo-Cp. Further research is needed to explain normal plasma iron in CuD mice. Reduction in Cp is a sensitive biomarker for copper deficiency. PMID:20170749

ArcS, the cognate sensor kinase in an atypical Arc system of Shewanella oneidensis MR-1.

PubMed

Lassak, Jürgen; Henche, Anna-Lena; Binnenkade, Lucas; Thormann, Kai M

2010-05-01

The availability of oxygen is a major environmental factor for many microbes, in particular for bacteria such as Shewanella species, which thrive in redox-stratified environments. One of the best-studied systems involved in mediating the response to changes in environmental oxygen levels is the Arc two-component system of Escherichia coli, consisting of the sensor kinase ArcB and the cognate response regulator ArcA. An ArcA ortholog was previously identified in Shewanella, and as in Escherichia coli, Shewanella ArcA is involved in regulating the response to shifts in oxygen levels. Here, we identified the hybrid sensor kinase SO_0577, now designated ArcS, as the previously elusive cognate sensor kinase of the Arc system in Shewanella oneidensis MR-1. Phenotypic mutant characterization, transcriptomic analysis, protein-protein interaction, and phosphotransfer studies revealed that the Shewanella Arc system consists of the sensor kinase ArcS, the single phosphotransfer domain protein HptA, and the response regulator ArcA. Phylogenetic analyses suggest that HptA might be a relict of ArcB. Conversely, ArcS is substantially different with respect to overall sequence homologies and domain organizations. Thus, we speculate that ArcS might have adopted the role of ArcB after a loss of the original sensor kinase, perhaps as a consequence of regulatory adaptation to a redox-stratified environment.

Fiber type- and fatty acid composition-dependent effects of high-fat diets on rat muscle triacylglyceride and fatty acid transporter protein-1 content.

PubMed

Marotta, Mario; Ferrer-Martnez, Andreu; Parnau, Josep; Turini, Marco; Macé, Katherine; Gómez Foix, Anna M

2004-08-01

Intramuscular triacylglyceride (TAG) is considered an independent marker of insulin resistance in humans. Here, we examined the effect of high-fat diets, based on distinct fatty acid compositions (saturated, monounsaturated or n-6 polyunsaturated), on TAG levels and fatty acid transporter protein (FATP-1) expression in 2 rat muscles that differ in their fiber type, soleus, and gastrocnemius; the relationship to whole body glucose intolerance was also studied. Compared with carbohydrate-fed rats, the groups subjected to any one of the high-fat diets consistently exhibited enhanced body weight gain and adiposity, elevated plasma free fatty acids and TAG in the fed condition, hyperinsulinemia, and glucose intolerance. TAG content was consistently higher in soleus than in gastrocnemius, but was only significantly elevated by the n-6 polyunsaturated-based diet. FATP-1 levels in soleus were double those in gastrocnemius muscle in carbohydrate-fed animals. High-fat diets caused an elevation in FATP-1 protein content in soleus, but a reduction in gastrocnemius. In conclusion, the hyperinsulinemic hyperlipidemic condition upregulates FATP-1 expression in soleus and downregulates that of gastrocnemius. Hypercaloric saturated, monounsaturated, or n-6 polyunsaturated lipid diets cause equivalent whole body insulin resistance in rats, but only an n-6 polyunsaturated acid-based diet triggers intramuscular TAG accumulation. Copyright 2004 Elsevier Inc.

Transcriptional Response of Honey Bee Larvae Infected with the Bacterial Pathogen Paenibacillus larvae

PubMed Central

Cornman, Robert Scott; Lopez, Dawn; Evans, Jay D.

2013-01-01

American foulbrood disease of honey bees is caused by the bacterium Paenibacillus larvae. Infection occurs per os in larvae and systemic infection requires a breaching of the host peritrophic matrix and midgut epithelium. Genetic variation exists for both bacterial virulence and host resistance, and a general immunity is achieved by larvae as they age, the basis of which has not been identified. To quickly identify a pool of candidate genes responsive to P. larvae infection, we sequenced transcripts from larvae inoculated with P. larvae at 12 hours post-emergence and incubated for 72 hours, and compared expression levels to a control cohort. We identified 75 genes with significantly higher expression and six genes with significantly lower expression. In addition to several antimicrobial peptides, two genes encoding peritrophic-matrix domains were also up-regulated. Extracellular matrix proteins, proteases/protease inhibitors, and members of the Osiris gene family were prevalent among differentially regulated genes. However, analysis of Drosophila homologs of differentially expressed genes revealed spatial and temporal patterns consistent with developmental asynchrony as a likely confounder of our results. We therefore used qPCR to measure the consistency of gene expression changes for a subset of differentially expressed genes. A replicate experiment sampled at both 48 and 72 hours post infection allowed further discrimination of genes likely to be involved in host response. The consistently responsive genes in our test set included a hymenopteran-specific protein tyrosine kinase, a hymenopteran specific serine endopeptidase, a cytochrome P450 (CYP9Q1), and a homolog of trynity, a zona pellucida domain protein. Of the known honey bee antimicrobial peptides, apidaecin was responsive at both time-points studied whereas hymenoptaecin was more consistent in its level of change between biological replicates and had the greatest increase in expression by RNA-seq analysis. PMID:23762370

Molecular dysexpression in gastric cancer revealed by integrated analysis of transcriptome data.

PubMed

Li, Xiaomei; Dong, Weiwei; Qu, Xueling; Zhao, Huixia; Wang, Shuo; Hao, Yixin; Li, Qiuwen; Zhu, Jianhua; Ye, Min; Xiao, Wenhua

2017-05-01

Gastric cancer (GC) is often diagnosed in the advanced stages and is associated with a poor prognosis. Obtaining an in depth understanding of the molecular mechanisms of GC has lagged behind compared with other cancers. This study aimed to identify candidate biomarkers for GC. An integrated analysis of microarray datasets was performed to identify differentially expressed genes (DEGs) between GC and normal tissues. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were then performed to identify the functions of the DEGs. Furthermore, a protein-protein interaction (PPI) network of the DEGs was constructed. The expression levels of the DEGs were validated in human GC tissues using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A set of 689 DEGs were identified in GC tissues, as compared with normal tissues, including 202 upregulated DEGs and 487 downregulated DEGs. The KEGG pathway analysis suggested that various pathways may play important roles in the pathology of GC, including pathways related to protein digestion and absorption, extracellular matrix-receptor interaction, and the metabolism of xenobiotics by cytochrome P450. The PPI network analysis indicated that the significant hub proteins consisted of SPP1, TOP2A and ARPC1B. RT-qPCR validation indicated that the expression levels of the top 10 most significantly dysexpressed genes were consistent with the illustration of the integrated analysis. The present study yielded a reference list of reliable DEGs, which represents a robust pool of candidates for further evaluation of GC pathogenesis and treatment.

A model of canopy photosynthesis incorporating protein distribution through the canopy and its acclimation to light, temperature and CO2

PubMed Central

Johnson, Ian R.; Thornley, John H. M.; Frantz, Jonathan M.; Bugbee, Bruce

2010-01-01

Background and Aims The distribution of photosynthetic enzymes, or nitrogen, through the canopy affects canopy photosynthesis, as well as plant quality and nitrogen demand. Most canopy photosynthesis models assume an exponential distribution of nitrogen, or protein, through the canopy, although this is rarely consistent with experimental observation. Previous optimization schemes to derive the nitrogen distribution through the canopy generally focus on the distribution of a fixed amount of total nitrogen, which fails to account for the variation in both the actual quantity of nitrogen in response to environmental conditions and the interaction of photosynthesis and respiration at similar levels of complexity. Model A model of canopy photosynthesis is presented for C3 and C4 canopies that considers a balanced approach between photosynthesis and respiration as well as plant carbon partitioning. Protein distribution is related to irradiance in the canopy by a flexible equation for which the exponential distribution is a special case. The model is designed to be simple to parameterize for crop, pasture and ecosystem studies. The amount and distribution of protein that maximizes canopy net photosynthesis is calculated. Key Results The optimum protein distribution is not exponential, but is quite linear near the top of the canopy, which is consistent with experimental observations. The overall concentration within the canopy is dependent on environmental conditions, including the distribution of direct and diffuse components of irradiance. Conclusions The widely used exponential distribution of nitrogen or protein through the canopy is generally inappropriate. The model derives the optimum distribution with characteristics that are consistent with observation, so overcoming limitations of using the exponential distribution. Although canopies may not always operate at an optimum, optimization analysis provides valuable insight into plant acclimation to environmental conditions. Protein distribution has implications for the prediction of carbon assimilation, plant quality and nitrogen demand. PMID:20861273

Improving protein fold recognition by extracting fold-specific features from predicted residue-residue contacts.

PubMed

Zhu, Jianwei; Zhang, Haicang; Li, Shuai Cheng; Wang, Chao; Kong, Lupeng; Sun, Shiwei; Zheng, Wei-Mou; Bu, Dongbo

2017-12-01

Accurate recognition of protein fold types is a key step for template-based prediction of protein structures. The existing approaches to fold recognition mainly exploit the features derived from alignments of query protein against templates. These approaches have been shown to be successful for fold recognition at family level, but usually failed at superfamily/fold levels. To overcome this limitation, one of the key points is to explore more structurally informative features of proteins. Although residue-residue contacts carry abundant structural information, how to thoroughly exploit these information for fold recognition still remains a challenge. In this study, we present an approach (called DeepFR) to improve fold recognition at superfamily/fold levels. The basic idea of our approach is to extract fold-specific features from predicted residue-residue contacts of proteins using deep convolutional neural network (DCNN) technique. Based on these fold-specific features, we calculated similarity between query protein and templates, and then assigned query protein with fold type of the most similar template. DCNN has showed excellent performance in image feature extraction and image recognition; the rational underlying the application of DCNN for fold recognition is that contact likelihood maps are essentially analogy to images, as they both display compositional hierarchy. Experimental results on the LINDAHL dataset suggest that even using the extracted fold-specific features alone, our approach achieved success rate comparable to the state-of-the-art approaches. When further combining these features with traditional alignment-related features, the success rate of our approach increased to 92.3%, 82.5% and 78.8% at family, superfamily and fold levels, respectively, which is about 18% higher than the state-of-the-art approach at fold level, 6% higher at superfamily level and 1% higher at family level. An independent assessment on SCOP_TEST dataset showed consistent performance improvement, indicating robustness of our approach. Furthermore, bi-clustering results of the extracted features are compatible with fold hierarchy of proteins, implying that these features are fold-specific. Together, these results suggest that the features extracted from predicted contacts are orthogonal to alignment-related features, and the combination of them could greatly facilitate fold recognition at superfamily/fold levels and template-based prediction of protein structures. Source code of DeepFR is freely available through https://github.com/zhujianwei31415/deepfr, and a web server is available through http://protein.ict.ac.cn/deepfr. zheng@itp.ac.cn or dbu@ict.ac.cn. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Overexpression of SERBP1 (Plasminogen activator inhibitor 1 RNA binding protein) in human breast cancer is correlated with favourable prognosis.

PubMed

Serce, Nuran Bektas; Boesl, Andreas; Klaman, Irina; von Serényi, Sonja; Noetzel, Erik; Press, Michael F; Dimmler, Arno; Hartmann, Arndt; Sehouli, Jalid; Knuechel, Ruth; Beckmann, Matthias W; Fasching, Peter A; Dahl, Edgar

2012-12-13

Plasminogen activator inhibitor 1 (PAI-1) overexpression is an important prognostic and predictive biomarker in human breast cancer. SERBP1, a protein that is supposed to regulate the stability of PAI-1 mRNA, may play a role in gynaecological cancers as well, since upregulation of SERBP1 was described in ovarian cancer recently. This is the first study to present a systematic characterisation of SERBP1 expression in human breast cancer and normal breast tissue at both the mRNA and the protein level. Using semiquantitative realtime PCR we analysed SERBP1 expression in different normal human tissues (n = 25), and in matched pairs of normal (n = 7) and cancerous breast tissues (n = 7). SERBP1 protein expression was analysed in two independent cohorts on tissue microarrays (TMAs), an initial evaluation set, consisting of 193 breast carcinomas and 48 normal breast tissues, and a second large validation set, consisting of 605 breast carcinomas. In addition, a collection of benign (n = 2) and malignant (n = 6) mammary cell lines as well as breast carcinoma lysates (n = 16) were investigated for SERBP1 expression by Western blot analysis. Furthermore, applying non-radioisotopic in situ hybridisation a subset of normal (n = 10) and cancerous (n = 10) breast tissue specimens from the initial TMA were analysed for SERBP1 mRNA expression. SERBP1 is not differentially expressed in breast carcinoma compared to normal breast tissue, both at the RNA and protein level. However, recurrence-free survival analysis showed a significant correlation (P = 0.008) between abundant SERBP1 expression in breast carcinoma and favourable prognosis. Interestingly, overall survival analysis also displayed a tendency (P = 0.09) towards favourable prognosis when SERBP1 was overexpressed in breast cancer. The RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a novel breast tumour marker with prognostic significance. Its potential involvement in the plasminogen activator protease cascade warrants further investigation.

Quantitative Proteomics Analysis of the cAMP/Protein Kinase A Signaling Pathway

PubMed Central

2012-01-01

To define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we used a quantitative proteomics approach in studies of wild-type (WT) and kin- (PKA-null) S49 murine T lymphoma cells. We also compared the impact of endogenous increases in the level of cAMP [by forskolin (Fsk) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX)] or by a cAMP analogue (8-CPT-cAMP). We identified 1056 proteins in WT and kin- S49 cells and found that 8-CPT-cAMP and Fsk with IBMX produced differences in protein expression. WT S49 cells had a correlation coefficient of 0.41 between DNA microarray data and the proteomics analysis in cells incubated with 8-CPT-cAMP for 24 h and a correlation coefficient of 0.42 between the DNA microarray data obtained at 6 h and the changes in protein expression after incubation with 8-CPT-cAMP for 24 h. Glutathione reductase (Gsr) had a higher level of basal expression in kin- S49 cells than in WT cells. Consistent with this finding, kin- cells are less sensitive to cell killing and generation of malondialdehyde than are WT cells incubated with H2O2. Cyclic AMP acting via PKA thus has a broad impact on protein expression in mammalian cells, including in the regulation of Gsr and oxidative stress. PMID:23110364

Application of wide selected-ion monitoring data-independent acquisition to identify tomato fruit proteins regulated by the CUTIN DEFICIENT2 transcription factor

PubMed Central

Martin, Laetitia B. B.; Sherwood, Robert W.; Nicklay, Joshua J.; Yang, Yong; Muratore-Schroeder, Tara L.; Anderson, Elizabeth T.; Thannhauser, Theodore W.; Rose, Jocelyn K. C.; Zhang, Sheng

2017-01-01

We describe here the use of label-free wide selected-ion monitoring data-independent acquisition (WiSIM-DIA) to identify proteins that are involved in the formation of tomato (Solanum lycopersicum) fruit cuticles and that are regulated by the transcription factor CUTIN DEFICIENT2 (CD2). A spectral library consisting of 11 753 unique peptides, corresponding to 2338 tomato protein groups, was used and the DIA analysis was performed at the MS1 level utilizing narrow mass windows for extraction with Skyline 2.6 software. We identified a total of 1140 proteins, 67 of which had expression levels that differed significantly between the cd2 tomato mutant and the wild-type cultivar M82. Differentially expressed proteins including a key protein involved in cutin biosynthesis, were selected for validation by target SRM/MRM and by Western blot analysis. In addition to confirming a role for CD2 in regulating cuticle formation, the results also revealed that CD2 influences pathways associated with cell wall biology, anthocyanin biosynthesis, plant development, and responses to stress, which complements findings of earlier RNA-Seq experiments. Our results provide new insights into molecular processes and aspects of fruit biology associated with CD2 function, and demonstrate that the WiSIM-DIA is an effective quantitative approach for global protein identifications. PMID:27089858

AML1/ETO induces self-renewal in hematopoietic progenitor cells via the Groucho-related amino-terminal AES protein.

PubMed

Steffen, Björn; Knop, Markus; Bergholz, Ulla; Vakhrusheva, Olesya; Rode, Miriam; Köhler, Gabriele; Henrichs, Marcel-Philipp; Bulk, Etmar; Hehn, Sina; Stehling, Martin; Dugas, Martin; Bäumer, Nicole; Tschanter, Petra; Brandts, Christian; Koschmieder, Steffen; Berdel, Wolfgang E; Serve, Hubert; Stocking, Carol; Müller-Tidow, Carsten

2011-04-21

The most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein, which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis, we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells, as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients, even in the absence of t(8;21). On a functional level, knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly, self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies, serial replating capacity of primary cells, and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML.

Markedly Lower Glutamic Acid Decarboxylase 67 Protein Levels in a Subset of Boutons in Schizophrenia.

PubMed

Rocco, Brad R; Lewis, David A; Fish, Kenneth N

2016-06-15

Convergent findings indicate that cortical gamma-aminobutyric acid (GABA)ergic circuitry is altered in schizophrenia. Postmortem studies have consistently found lower levels of glutamic acid decarboxylase 67 (GAD67) messenger RNA (mRNA) in the prefrontal cortex (PFC) of subjects with schizophrenia. At the cellular level, the density of GABA neurons with detectable levels of GAD67 mRNA is ~30% lower across cortical layers. Knowing how this transcript deficit translates to GAD67 protein levels in axonal boutons is important for understanding the impact it might have on GABA synthesis. In addition, because reductions in GAD67 expression before, but not after, the maturation of GABAergic boutons results in a lower density of GABAergic boutons in mouse cortical cultures, knowing if GABAergic bouton density is altered in schizophrenia would provide insight into the timing of the GAD67 deficit. PFC tissue sections from 20 matched pairs of schizophrenia and comparison subjects were immunolabeled for the vesicular GABA transporter (vGAT) and GAD67. vGAT+ bouton density did not differ between subject groups, consistent with findings that vGAT mRNA levels are unaltered in the illness and confirming that the number of cortical GABAergic boutons is not lower in schizophrenia. In contrast, in schizophrenia subjects, the proportion of vGAT+ boutons with detectable GAD67 levels (vGAT+/GAD67+ boutons) was 16% lower and mean GAD67 levels were 14% lower in the remaining vGAT+/GAD67+ boutons. Our findings suggest that GABA production is markedly reduced in a subset of boutons in the PFC of schizophrenia subjects and that this reduction likely occurs after the maturation of GABAergic boutons. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Pharmacokinetics, pharmacodynamics, and efficacy of a small-molecule SMN2 splicing modifier in mouse models of spinal muscular atrophy

PubMed Central

Zhao, Xin; Feng, Zhihua; Ling, Karen K. Y.; Mollin, Anna; Sheedy, Josephine; Yeh, Shirley; Petruska, Janet; Narasimhan, Jana; Dakka, Amal; Welch, Ellen M.; Karp, Gary; Chen, Karen S.; Metzger, Friedrich; Ratni, Hasane; Lotti, Francesco; Tisdale, Sarah; Naryshkin, Nikolai A.; Pellizzoni, Livio; Paushkin, Sergey; Ko, Chien-Ping; Weetall, Marla

2016-01-01

Spinal muscular atrophy (SMA) is caused by the loss or mutation of both copies of the survival motor neuron 1 (SMN1) gene. The related SMN2 gene is retained, but due to alternative splicing of exon 7, produces insufficient levels of the SMN protein. Here, we systematically characterize the pharmacokinetic and pharmacodynamics properties of the SMN splicing modifier SMN-C1. SMN-C1 is a low-molecular weight compound that promotes the inclusion of exon 7 and increases production of SMN protein in human cells and in two transgenic mouse models of SMA. Furthermore, increases in SMN protein levels in peripheral blood mononuclear cells and skin correlate with those in the central nervous system (CNS), indicating that a change of these levels in blood or skin can be used as a non-invasive surrogate to monitor increases of SMN protein levels in the CNS. Consistent with restored SMN function, SMN-C1 treatment increases the levels of spliceosomal and U7 small-nuclear RNAs and corrects RNA processing defects induced by SMN deficiency in the spinal cord of SMNΔ7 SMA mice. A 100% or greater increase in SMN protein in the CNS of SMNΔ7 SMA mice robustly improves the phenotype. Importantly, a ∼50% increase in SMN leads to long-term survival, but the SMA phenotype is only partially corrected, indicating that certain SMA disease manifestations may respond to treatment at lower doses. Overall, we provide important insights for the translation of pre-clinical data to the clinic and further therapeutic development of this series of molecules for SMA treatment. PMID:26931466

Identification of differentially expressed proteins of Arthrospira (Spirulina) plantensis-YZ under salt-stress conditions by proteomics and qRT-PCR analysis

PubMed Central

2013-01-01

Arthrospira (Spirulina) platensis as a representative species of cyanobacteria has been recognized and used worldwide as a source of protein in the food, which possesses some unusual and valuable physiological characteristics, such as alkali and salt tolerance. Based on complete genome sequencing of Arthrospira (Spirulina) plantensis-YZ, we compared the protein expression profiles of this organism under different salt-stress conditions (i.e. 0.02 M, 0.5 M and 1.0 M NaCl, respectively), using 2-D electrophoresis and peptide mass fingerprinting, and retrieved 141 proteins showing significantly differential expression in response to salt-stress. Of the 141 proteins, 114 Arthrospira (Spirulina) plantensis-YZ proteins were found with significant homology to those found in Arthrospira (76 proteins in Arthrospira platensis str. Paraca and 38 in Arthrospira maxima CS-328). The remaining 27 proteins belong to other bacteria. Subsequently, we determined the transcriptional level of 29 genes in vivo in response to NaCl treatments and verified them by qRT-PCR. We found that 12 genes keep consistency at both transcription and protein levels, and transcription of all of them but one were up-regulated. We classified the 141 differentially expressed proteins into 18 types of function categories using COG database, and linked them to their respective KEGG metabolism pathways. These proteins are involved in 31 metabolism pathways, such as photosynthesis, glucose metabolism, cysteine and methionine metabolism, lysine synthesis, fatty acid metabolism, glutathione metabolism. Additionally, the SRPs, heat shock protein and ABC transporter proteins were identified, which probably render Arthrospira (Spirulina) plantensis’s resistance against high salt stress. PMID:23363438

Expression of Zinc Finger and BTB Domain-containing 7A in Colorectal Carcinoma.

PubMed

Joo, Jin Woo; Kim, Hyun-Soo; Do, Sung-Im; Sung, Ji-Youn

2018-05-01

Previous studies have revealed that zinc finger and BTB domain-containing 7A (ZBTB7A), an important proto-oncogene, plays multiple roles in carcinogenesis and is up-regulated in several human malignancies. However, the expression of ZBTB7A in colorectal carcinoma (CRC) has seldom been documented. In this study, we investigated the differential expression of ZBTB7A in CRC cell lines and tissues. Expression levels of ZBTB7A mRNA and protein were examined in CRC cell lines. ZBTB7A protein expression was also evaluated in tissue samples of normal colonic mucosa, high-grade dysplasia, and CRC using immunohistochemical staining. All CRC cell lines exhibited significantly higher ZBTB7A mRNA expression levels than did normal colonic epithelial cells. The ZBTB7A protein expression levels were clearly higher in the CRC cell lines than in the normal colonic epithelial cells. Consistent with the cell line data, immunostaining revealed that there were significant differences in ZBTB7A protein expression between tissue samples of CRC and normal colonic mucosa (p=0.048) and high-grade dysplasia (p=0.015). In addition, metastatic CRC exhibited significantly higher ZBTB7A protein expression levels than primary CRC (p=0.027). We demonstrated that ZBTB7A expression is up-regulated in CRC cell lines and tissues. Our data suggest that ZBTB7A is involved in the development and progression of CRC. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice.

PubMed

Tomé, Stéphanie; Manley, Kevin; Simard, Jodie P; Clark, Greg W; Slean, Meghan M; Swami, Meera; Shelbourne, Peggy F; Tillier, Elisabeth R M; Monckton, Darren G; Messer, Anne; Pearson, Christopher E

2013-01-01

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.

MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice

PubMed Central

Simard, Jodie P.; Clark, Greg W.; Slean, Meghan M.; Swami, Meera; Shelbourne, Peggy F.; Tillier, Elisabeth R. M.; Monckton, Darren G.; Messer, Anne; Pearson, Christopher E.

2013-01-01

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases. PMID:23468640

The Redox Proteome*

PubMed Central

Go, Young-Mi; Jones, Dean P.

2013-01-01

The redox proteome consists of reversible and irreversible covalent modifications that link redox metabolism to biologic structure and function. These modifications, especially of Cys, function at the molecular level in protein folding and maturation, catalytic activity, signaling, and macromolecular interactions and at the macroscopic level in control of secretion and cell shape. Interaction of the redox proteome with redox-active chemicals is central to macromolecular structure, regulation, and signaling during the life cycle and has a central role in the tolerance and adaptability to diet and environmental challenges. PMID:23861437

Free fatty acid particles in protein formulations, part 2: contribution of polysorbate raw material.

PubMed

Siska, Christine C; Pierini, Christopher J; Lau, Hollis R; Latypov, Ramil F; Fesinmeyer, R Matthew; Litowski, Jennifer R

2015-02-01

Polysorbate 20 (PS20) is a nonionic surfactant frequently used to stabilize protein biopharmaceuticals. During the development of mAb formulations containing PS20, small clouds of particles were observed in solutions stored in vials. The degree of particle formation was dependent on PS20 concentration. The particles were characterized by reversed-phase HPLC after dissolution and labeling with the fluorescent dye 1-pyrenyldiazomethane. The analysis showed that the particles consisted of free fatty acids (FFAs), with the distribution of types consistent with those found in the PS20 raw material. Protein solutions formulated with polysorbate 80, a chemically similar nonionic surfactant, showed a substantial delay in particle formation over time compared with PS20. Multiple lots of polysorbates were evaluated for FFA levels, each exhibiting differences based on polysorbate type and lot. Polysorbates purchased in more recent years show a greater distribution and quantity of FFA and also a greater propensity to form particles. This work shows that the quality control of polysorbate raw materials could play an important role in biopharmaceutical product quality. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Apoptosis of enterocytes and nitration of junctional complex proteins promote alcohol-induced gut leakiness and liver injury.

PubMed

Cho, Young-Eun; Yu, Li-Rong; Abdelmegeed, Mohamed A; Yoo, Seong-Ho; Song, Byoung-Joon

2018-07-01

Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury, although the molecular mechanisms are still elusive. This study was aimed at investigating the roles of apoptosis of enterocytes and nitration followed by degradation of intestinal tight junction (TJ) and adherens junction (AJ) proteins in binge alcohol-induced gut leakiness. The levels of intestinal (ileum) junctional complex proteins, oxidative stress markers and apoptosis-related proteins in rodents, T84 colonic cells and autopsied human ileums were determined by immunoblot, immunoprecipitation, immunofluorescence, and mass-spectral analyses. Binge alcohol exposure caused apoptosis of gut enterocytes with elevated serum endotoxin and liver injury. The levels of intestinal CYP2E1, iNOS, nitrated proteins and apoptosis-related marker proteins were significantly elevated in binge alcohol-exposed rodents. Differential, quantitative mass-spectral analyses of the TJ-enriched fractions of intestinal epithelial layers revealed that several TJ, AJ and desmosome proteins were decreased in binge alcohol-exposed rats compared to controls. Consistently, the levels of TJ proteins (claudin-1, claudin-4, occludin and zonula occludens-1), AJ proteins (β-catenin and E-cadherin) and desmosome plakoglobin were very low in binge alcohol-exposed rats, wild-type mice, and autopsied human ileums but not in Cyp2e1-null mice. Additionally, pretreatment with specific inhibitors of CYP2E1 and iNOS prevented disorganization and/or degradation of TJ proteins in alcohol-exposed T84 colonic cells. Furthermore, immunoprecipitation followed by immunoblot confirmed that intestinal TJ and AJ proteins were nitrated and degraded via ubiquitin-dependent proteolysis, resulting in their decreased levels. These results demonstrated for the first time the critical roles of CYP2E1, apoptosis of enterocytes, and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins, in promoting binge alcohol-induced gut leakiness and endotoxemia, contributing to inflammatory liver disease. Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury. Our results demonstrated for the first time the critical roles of apoptosis of enterocytes and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins in promoting this gut leakiness and endotoxemia. These results provide insight into the molecular mechanisms of alcohol-induced inflammatory liver disease. Published by Elsevier B.V.

Myostatin induces insulin resistance via Casitas B-lineage lymphoma b (Cblb)-mediated degradation of insulin receptor substrate 1 (IRS1) protein in response to high calorie diet intake.

PubMed

Bonala, Sabeera; Lokireddy, Sudarsanareddy; McFarlane, Craig; Patnam, Sreekanth; Sharma, Mridula; Kambadur, Ravi

2014-03-14

To date a plethora of evidence has clearly demonstrated that continued high calorie intake leads to insulin resistance and type-2 diabetes with or without obesity. However, the necessary signals that initiate insulin resistance during high calorie intake remain largely unknown. Our results here show that in response to a regimen of high fat or high glucose diets, Mstn levels were induced in muscle and liver of mice. High glucose- or fat-mediated induction of Mstn was controlled at the level of transcription, as highly conserved carbohydrate response and sterol-responsive (E-box) elements were present in the Mstn promoter and were revealed to be critical for ChREBP (carbohydrate-responsive element-binding protein) or SREBP1c (sterol regulatory element-binding protein 1c) regulation of Mstn expression. Further molecular analysis suggested that the increased Mstn levels (due to high glucose or fatty acid loading) resulted in increased expression of Cblb in a Smad3-dependent manner. Casitas B-lineage lymphoma b (Cblb) is an ubiquitin E3 ligase that has been shown to specifically degrade insulin receptor substrate 1 (IRS1) protein. Consistent with this, our results revealed that elevated Mstn levels specifically up-regulated Cblb, resulting in enhanced ubiquitin proteasome-mediated degradation of IRS1. In addition, over expression or knock down of Cblb had a major impact on IRS1 and pAkt levels in the presence or absence of insulin. Collectively, these observations strongly suggest that increased glucose levels and high fat diet, both, result in increased circulatory Mstn levels. The increased Mstn in turn is a potent inducer of insulin resistance by degrading IRS1 protein via the E3 ligase, Cblb, in a Smad3-dependent manner.

Sensory perception of and salivary protein response to astringency as a function of the 6-n-propylthioural (PROP) bitter-taste phenotype.

PubMed

Melis, Melania; Yousaf, Neeta Y; Mattes, Mitchell Z; Cabras, Tiziana; Messana, Irene; Crnjar, Roberto; Tomassini Barbarossa, Iole; Tepper, Beverly J

2017-05-01

Individual differences in astringency perception are poorly understood. Astringency from tannins stimulates the release of specific classes of salivary proteins. These proteins form complexes with tannins, altering their perceived astringency and reducing their bioavailability. We studied the bitter compound, 6-n-propylthioural (PROP), as a phenotypic marker for variation in astringency perception and salivary protein responses. Seventy-nine subjects classified by PROP taster status rated cranberry juice cocktail (CJC; with added sugar) supplemented with 0, 1.5 or 2.0g/L tannic acid (TA). Saliva for protein analyses was collected at rest, or after stimulation with TA or cranberry juice (CJ; without added sugar). CJC with 1.5g/L tannic acid was found to be less astringent, and was liked more by PROP non-taster males than PROP taster males, consistent with the expectation that non-tasters are less sensitive to astringency. Levels of acidic Proline Rich Proteins (aPRPs) and basic Proline Rich Proteins (bPRPs) decreased after TA, while levels of aPRPs, bPRPs and Cystatins unexpectedly rose after CJ. Increases in bPRPs and Cystatins were only observed in PROP tasters. The PROP phenotype plays a gender-specific, but somewhat limited role in the perceived astringency of tannic-acid supplemented, cranberry juice cocktail. The PROP phenotype (regardless of gender) may also be involved in the release of salivary proteins previously implicated in oral health. Copyright © 2017 Elsevier Inc. All rights reserved.

Protein modification in the post-mating spermatophore of the signal crayfish Pacifastacus leniusculus: insight into the tyrosine phosphorylation in a non-motile spermatozoon.

PubMed

Niksirat, Hamid; Vancová, Marie; Andersson, Liselotte; James, Peter; Kouba, Antonín; Kozák, Pavel

2016-09-01

After mating, spermatophores of signal crayfish are stored on the body of the female for a period before fertilization. This study compared the post-mating protein profile and pattern of protein tyrosine phosphorylation of the signal crayfish spermatophore to that of the freshly ejaculated spermatophore and found substantial differences. Two major bands of tyrosine-phosphorylated proteins of molecular weights 10 and 50kDa were observed in the freshly ejaculated spermatophore of the signal crayfish. While the tyrosine-phosphorylated protein band with molecular weight 10kDa was formed by protein(s) of similar pH, the band with molecular weight of 50kDa consisted of proteins of varying pH. In the post-mating spermatophore, the band with molecular weight of 50kDa was not detected, and an increase in the level of protein tyrosine phosphorylation was observed in the 10kDa band. The microtubular radial arms of the spermatozoon showed a positive reaction to an anti-tyrosine antibody conjugated with gold particles in both the freshly ejaculated and post-mating spermatophores. In conclusion, the male gamete of the signal crayfish undergoes molecular modification during post-mating storage on the body of the female including changes in the level of protein expression and protein tyrosine phosphorylation. Structural similarity of the radial arms in the crayfish immotile spermatozoon with flagellum, which is the main site of protein tyrosine phosphorylation in the mammalian motile spermatozoa, raises questions regarding evolution and function of such organelles across the animal kingdom that must be addressed in the future studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of SLEEPLESS, a sleep-promoting factor.

PubMed

Koh, Kyunghee; Joiner, William J; Wu, Mark N; Yue, Zhifeng; Smith, Corinne J; Sehgal, Amita

2008-07-18

Sleep is an essential process conserved from flies to humans. The importance of sleep is underscored by its tight homeostatic control. Through a forward genetic screen, we identified a gene, sleepless, required for sleep in Drosophila. The sleepless gene encodes a brain-enriched, glycosylphosphatidylinositol-anchored protein. Loss of SLEEPLESS protein caused an extreme (>80%) reduction in sleep; a moderate reduction in SLEEPLESS had minimal effects on baseline sleep but markedly reduced the amount of recovery sleep after sleep deprivation. Genetic and molecular analyses revealed that quiver, a mutation that impairs Shaker-dependent potassium current, is an allele of sleepless. Consistent with this finding, Shaker protein levels were reduced in sleepless mutants. We propose that SLEEPLESS is a signaling molecule that connects sleep drive to lowered membrane excitability.

Protection of Cattle against Foot-and-Mouth Disease by a Synthetic Peptide

NASA Astrophysics Data System (ADS)

Dimarchi, Richard; Brooke, Gerald; Gale, Charles; Cracknell, Victor; Doel, Timothy; Mowat, Noel

1986-05-01

A chemically synthesized peptide consisting essentially of two separate regions (residues 141 to 158 and 200 to 213) of a virus coat protein (VP1) from the 01 Kaufbeuren strain of foot-and-mouth disease virus was prepared free of any carrier protein. It elicited high levels of neutralizing antibody and protected cattle against intradermolingual challenge by inoculation with infectious virus. Comparative evaluation of this peptide with a single-site peptide (residues 141 to 158) in guinea pigs suggests the importance of the VP1 carboxyl terminal residues in enhancing the protective response.

3Drefine: an interactive web server for efficient protein structure refinement.

PubMed

Bhattacharya, Debswapna; Nowotny, Jackson; Cao, Renzhi; Cheng, Jianlin

2016-07-08

3Drefine is an interactive web server for consistent and computationally efficient protein structure refinement with the capability to perform web-based statistical and visual analysis. The 3Drefine refinement protocol utilizes iterative optimization of hydrogen bonding network combined with atomic-level energy minimization on the optimized model using a composite physics and knowledge-based force fields for efficient protein structure refinement. The method has been extensively evaluated on blind CASP experiments as well as on large-scale and diverse benchmark datasets and exhibits consistent improvement over the initial structure in both global and local structural quality measures. The 3Drefine web server allows for convenient protein structure refinement through a text or file input submission, email notification, provided example submission and is freely available without any registration requirement. The server also provides comprehensive analysis of submissions through various energy and statistical feedback and interactive visualization of multiple refined models through the JSmol applet that is equipped with numerous protein model analysis tools. The web server has been extensively tested and used by many users. As a result, the 3Drefine web server conveniently provides a useful tool easily accessible to the community. The 3Drefine web server has been made publicly available at the URL: http://sysbio.rnet.missouri.edu/3Drefine/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The human chromokinesin Kid is a plus end-directed microtubule-based motor

PubMed Central

Yajima, Junichiro; Edamatsu, Masaki; Watai-Nishii, Junko; Tokai-Nishizumi, Noriko; Yamamoto, Tadashi; Toyoshima, Yoko Y.

2003-01-01

Kid is a kinesin-like DNA-binding protein known to be involved in chromosome movement during mitosis, although its actual motor function has not been demonstrated. Here, we describe the initial characterization of Kid as a microtubule-based motor using optical trapping microscopy. A bacterially expressed fusion protein consisting of a truncated Kid fragment (amino acids 1–388 or 1–439) is indeed an active microtubule motor with an average speed of ∼160 nm/s, and the polarity of movement is plus end directed. We could not detect processive movement of either monomeric Kid or dimerizing chimeric Kid; however, low levels of processivity (a few steps) cannot be detected with our method. These results are consistent with Kid having a role in chromosome congression in vivo, where it would be responsible for the polar ejection forces acting on the chromosome arms. PMID:12606572

Dietary protein intake affects expression of genes for lipid metabolism in porcine skeletal muscle in a genotype-dependent manner.

PubMed

Liu, Yingying; Li, Fengna; He, Lingyun; Tan, Bie; Deng, Jinping; Kong, Xiangfeng; Li, Yinghui; Geng, Meimei; Yin, Yulong; Wu, Guoyao

2015-04-14

Skeletal muscle is a major site for the oxidation of fatty acids (FA) in mammals, including humans. Using a swine model, we tested the hypothesis that dietary protein intake regulates the expression of key genes for lipid metabolism in skeletal muscle. A total of ninety-six barrows (forty-eight pure-bred Bama mini-pigs (fatty genotype) and forty-eight Landrace pigs (lean genotype)) were fed from 5 weeks of age to market weight. Pigs of fatty or lean genotype were randomly assigned to one of two dietary treatments (low- or adequate-protein diet), with twenty-four individually fed pigs per treatment. Our data showed that dietary protein levels affected the expression of genes involved in the anabolism and catabolism of lipids in the longissimus dorsi and biceps femoris muscles in a genotype-dependent manner. Specifically, Bama mini-pigs had more intramuscular fat, SFA and MUFA, as well as elevated mRNA expression levels of lipogenic genes, compared with Landrace pigs. In contrast, Bama mini-pigs had lower mRNA expression levels of lipolytic genes than Landrace pigs fed an adequate-protein diet in the growing phase. These data are consistent with higher white-fat deposition in Bama mini-pigs than in Landrace pigs. In conclusion, adequate provision of dietary protein (amino acids) plays an important role in regulating the expression of key lipogenic genes, and the growth of white adipose tissue, in a genotype- and tissue-specific manner. These findings have important implications for developing novel dietary strategies in pig production.

Biomarker Discovery and Verification of Esophageal Squamous Cell Carcinoma Using Integration of SWATH/MRM.

PubMed

Hou, Guixue; Lou, Xiaomin; Sun, Yulin; Xu, Shaohang; Zi, Jin; Wang, Quanhui; Zhou, Baojin; Han, Bo; Wu, Lin; Zhao, Xiaohang; Lin, Liang; Liu, Siqi

2015-09-04

We propose an efficient integration of SWATH with MRM for biomarker discovery and verification when the corresponding ion library is well established. We strictly controlled the false positive rate associated with SWATH MS signals and carefully selected the target peptides coupled with SWATH and MRM. We collected 10 samples of esophageal squamous cell carcinoma (ESCC) tissues paired with tumors and adjacent regions and quantified 1758 unique proteins with FDR 1% at protein level using SWATH, in which 467 proteins were abundance-dependent with ESCC. After carefully evaluating the SWATH MS signals of the up-regulated proteins, we selected 120 proteins for MRM verification. MRM analysis of the pooled and individual esophageal tissues resulted in 116 proteins that exhibited similar abundance response modes to ESCC that were acquired with SWATH. Because the ESCC-related proteins consisted of a high percentile of secreted proteins, we conducted the MRM assay on patient sera that were collected from pre- and postoperation. Of the 116 target proteins, 42 were identified in the ESCC sera, including 11 with lowered abundances postoperation. Coupling SWATH and MRM is thus feasible and efficient for the discovery and verification of cancer-related protein biomarkers.

Abundance of Plasma Antioxidant Proteins Confers Tolerance to Acute Hypobaric Hypoxia Exposure

PubMed Central

Padhy, Gayatri; Sethy, Niroj Kumar; Ganju, Lilly

2013-01-01

Abstract Padhy, Gayatri, Niroj Kumar Sethy, Lilly Ganju, and Kalpana Bhargava. Abundance of plasma antioxidant proteins confers tolerance to acute hypobaric hypoxia exposure. High Alt Med Biol 14:289–297, 2013—Systematic identification of molecular signatures for hypobaric hypoxia can aid in better understanding of human adaptation to high altitude. In an attempt to identify proteins promoting hypoxia tolerance during acute exposure to high altitude, we screened and identified hypoxia tolerant and susceptible rats based on hyperventilation time to a simulated altitude of 32,000 ft (9754 m). The hypoxia tolerance was further validated by estimating 8-isoprotane levels and protein carbonyls, which revealed that hypoxia tolerant rats possessed significant lower plasma levels as compared to susceptible rats. We used a comparative plasma proteome profiling approach using 2-dimensional gel electrophoresis (2-DGE) combined with MALDI TOF/TOF for both groups, along with an hypoxic control group. This resulted in the identification of 19 differentially expressed proteins. Seven proteins (TTR, GPx-3, PON1, Rab-3D, CLC11, CRP, and Hp) were upregulated in hypoxia tolerant rats, while apolipoprotein A-I (APOA1) was upregulated in hypoxia susceptible rats. We further confirmed the consistent higher expression levels of three antioxidant proteins (PON1, TTR, and GPx-3) in hypoxia-tolerant animals using ELISA and immunoblotting. Collectively, these proteomics-based results highlight the role of antioxidant enzymes in conferring hypoxia tolerance during acute hypobaric hypoxia. The expression of these antioxidant enzymes could be used as putative biomarkers for screening altitude adaptation as well as aiding in better management of altered oxygen pathophysiologies. PMID:24067188

Assessing Nutritional Parameters of Brown Bear Diets among Ecosystems Gives Insight into Differences among Populations.

PubMed

López-Alfaro, Claudia; Coogan, Sean C P; Robbins, Charles T; Fortin, Jennifer K; Nielsen, Scott E

2015-01-01

Food habit studies are among the first steps used to understand wildlife-habitat relationships. However, these studies are in themselves insufficient to understand differences in population productivity and life histories, because they do not provide a direct measure of the energetic value or nutritional composition of the complete diet. Here, we developed a dynamic model integrating food habits and nutritional information to assess nutritional parameters of brown bear (Ursus arctos) diets among three interior ecosystems of North America. Specifically, we estimate the average amount of digestible energy and protein (per kilogram fresh diet) content in the diet and across the active season by bears living in western Alberta, the Flathead River (FR) drainage of southeast British Columbia, and the Greater Yellowstone Ecosystem (GYE). As well, we estimate the proportion of energy and protein in the diet contributed by different food items, thereby highlighting important food resources in each ecosystem. Bear diets in Alberta had the lowest levels of digestible protein and energy through all seasons, which might help explain the low reproductive rates of this population. The FR diet had protein levels similar to the recent male diet in the GYE during spring, but energy levels were lower during late summer and fall. Historic and recent diets in GYE had the most energy and protein, which is consistent with their larger body sizes and higher population productivity. However, a recent decrease in consumption of trout (Oncorhynchus clarki), whitebark pine nuts (Pinus albicaulis), and ungulates, particularly elk (Cervus elaphus), in GYE bears has decreased the energy and protein content of their diet. The patterns observed suggest that bear body size and population densities are influenced by seasonal availability of protein an energy, likely due in part to nutritional influences on mass gain and reproductive success.

Assessing Nutritional Parameters of Brown Bear Diets among Ecosystems Gives Insight into Differences among Populations

PubMed Central

López-Alfaro, Claudia; Coogan, Sean C. P.; Robbins, Charles T.; Fortin, Jennifer K.; Nielsen, Scott E.

2015-01-01

Food habit studies are among the first steps used to understand wildlife-habitat relationships. However, these studies are in themselves insufficient to understand differences in population productivity and life histories, because they do not provide a direct measure of the energetic value or nutritional composition of the complete diet. Here, we developed a dynamic model integrating food habits and nutritional information to assess nutritional parameters of brown bear (Ursus arctos) diets among three interior ecosystems of North America. Specifically, we estimate the average amount of digestible energy and protein (per kilogram fresh diet) content in the diet and across the active season by bears living in western Alberta, the Flathead River (FR) drainage of southeast British Columbia, and the Greater Yellowstone Ecosystem (GYE). As well, we estimate the proportion of energy and protein in the diet contributed by different food items, thereby highlighting important food resources in each ecosystem. Bear diets in Alberta had the lowest levels of digestible protein and energy through all seasons, which might help explain the low reproductive rates of this population. The FR diet had protein levels similar to the recent male diet in the GYE during spring, but energy levels were lower during late summer and fall. Historic and recent diets in GYE had the most energy and protein, which is consistent with their larger body sizes and higher population productivity. However, a recent decrease in consumption of trout (Oncorhynchus clarki), whitebark pine nuts (Pinus albicaulis), and ungulates, particularly elk (Cervus elaphus), in GYE bears has decreased the energy and protein content of their diet. The patterns observed suggest that bear body size and population densities are influenced by seasonal availability of protein an energy, likely due in part to nutritional influences on mass gain and reproductive success. PMID:26083536

BLOOD PLASMA PROTEIN REGENERATION CONTROLLED BY DIET

PubMed Central

Holman, Russell L.; Mahoney, Earle B.; Whipple, George H.

1934-01-01

When blood plasma proteins are depleted by bleeding and return of the washed red cells (plasmapheresis) the regeneration of new plasma proteins can be controlled at will by diet. The amount and character of protein intake is all important. Liver protein and casein are efficient proteins to promote rapid regeneration of plasma proteins but some vegetable proteins are also efficient. The blood plasma proteins are reduced by plasmapheresis close to the edema level (3.5–4.0 per cent) and kept at this level by suitable exchanges almost daily. The amount of plasma protein removed is credited to the given diet period. A basal ration is used which is poor in vegetable protein (potato) and contains no animal protein. The dog on this ration can be kept in nitrogen balance but can produce only about 2 gm. plasma protein per kilo body weight per week. With liver or casein feeding this production can be increased three- or fourfold. A reserve of protein building material can be demonstrated in the normal dog when its plasma proteins are depleted. In the first 3 weeks of depletion this reserve in excess of the final basal output may amount to 3–20 gm. protein. This may be stored at least in part in the liver. As much as 50 per cent of this reserve may be albumin or albumin producing material. A reversal of the albumin-globulin ratio may be observed on the basal diet alone. The reversal will always follow plasmapheresis with the dog on the basal diet and the total plasma protein output will consist approximately of 2 parts globulin and 1 part albumin. Liver diet will raise the production and output of albumin and bring the ratio back toward normal. Albumin production may actually exceed the globulin output during liver diet periods. The change is less conspicuous with casein but in the same direction. PMID:19870244

Self-regulation of 70-kilodalton heat shock proteins in Saccharomyces cerevisiae.

PubMed Central

Stone, D E; Craig, E A

1990-01-01

To determine whether the 70-kilodalton heat shock proteins of Saccharomyces cerevisiae play a role in regulating their own synthesis, we studied the effect of overexpressing the SSA1 protein on the activity of the SSA1 5'-regulatory region. The constitutive level of Ssa1p was increased by fusing the SSA1 structural gene to the GAL1 promoter. A reporter vector consisting of an SSA1-lacZ translational fusion was used to assess SSA1 promoter activity. In a strain producing approximately 10-fold the normal heat shock level of Ssa1p, induction of beta-galactosidase activity by heat shock was almost entirely blocked. Expression of a transcriptional fusion vector in which the CYC1 upstream activating sequence of a CYC1-lacZ chimera was replaced by a sequence containing a heat shock upstream activating sequence (heat shock element 2) from the 5'-regulatory region of SSA1 was inhibited by excess Ssa1p. The repression of an SSA1 upstream activating sequence by the SSA1 protein indicates that SSA1 self-regulation is at least partially mediated at the transcriptional level. The expression of another transcriptional fusion vector, containing heat shock element 2 and a lesser amount of flanking sequence, is not inhibited when Ssa1p is overexpressed. This suggests the existence of an element, proximal to or overlapping heat shock element 2, that confers sensitivity to the SSA1 protein. Images PMID:2181281

Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein, TkSRPP3, result in qualitative and quantitative changes in rubber metabolism.

PubMed

Collins-Silva, Jillian; Nural, Aise Taban; Skaggs, Amanda; Scott, Deborah; Hathwaik, Upul; Woolsey, Rebekah; Schegg, Kathleen; McMahan, Colleen; Whalen, Maureen; Cornish, Katrina; Shintani, David

2012-07-01

Several proteins have been identified and implicated in natural rubber biosynthesis, one of which, the small rubber particle protein (SRPP), was originally identified in Hevea brasiliensis as an abundant protein associated with cytosolic vesicles known as rubber particles. While previous in vitro studies suggest that SRPP plays a role in rubber biosynthesis, in vivo evidence is lacking to support this hypothesis. To address this issue, a transgene approach was taken in Taraxacum kok-saghyz (Russian dandelion or Tk) to determine if altered SRPP levels would influence rubber biosynthesis. Three dandelion SRPPs were found to be highly abundant on dandelion rubber particles. The most abundant particle associated SRPP, TkSRPP3, showed temporal and spatial patterns of expression consistent with patterns of natural rubber accumulation in dandelion. To confirm its role in rubber biosynthesis, TkSRPP3 expression was altered in Russian dandelion using over-expression and RNAi methods. While TkSRPP3 over-expressing lines had slightly higher levels of rubber in their roots, relative to the control, TkSRPP3 RNAi lines showed significant decreases in root rubber content and produced dramatically lower molecular weight rubber than the control line. Not only do results here provide in vivo evidence of TkSRPP proteins affecting the amount of rubber in dandelion root, but they also suggest a function in regulating the molecular weight of the cis-1, 4-polyisoprene polymer. Copyright © 2012 Elsevier Ltd. All rights reserved.

Possible molecular mechanism underlying cadmium-induced circadian rhythms disruption in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Bo; Chen, Tian-Ming; Zhong, Yingbin

This study was aimed to explore the mechanisms underlying cadmium-induced circadian rhythms disruption. Two groups of zebrafish larvae treated with or without 5 ppm CdCl{sub 2} were incubated in a photoperiod of 14-h light/10-h dark conditions. The mRNA levels of clock1a, bmal1b, per2 and per1b in two groups were determined. Microarray data were generated in two group of samples. Differential expression of genes were identified and the changes in expression level for some genes were validated by RT-PCR. Finally, Gene Ontology functional and KEGG pathway enrichment analysis of differentially expressed genes (DEGs) were performed. In comparison with normal group, the mRNAmore » levels of clock1a, bmal1b, and per2 were significantly changed and varied over the circadian cycle in CdCl2-treated group. DEGs were obtained from the light (84 h, ZT12) and dark (88 h, ZT16) phase. In addition, G-protein coupled receptor protein signaling pathway and immune response were both enriched by DEGs in both groups. While, proteolysis and amino acid metabolism were found associated with DEGs in light phase, and Neuroactive ligand-receptor interaction and oxidation-reduction process were significantly enriched by DEGs in dark phase. Besides, the expression pattern of genes including hsp70l and or115-11 obtained by RT-PCR were consistent with those obtained by microarray analysis. As a consequence, cadmium could make significant effects on circadian rhythms through immune response and G protein-coupled receptor signaling pathway. Besides, between the dark and the light phase, the mechanism by which cadmium inducing disruption of circadian rhythms were different to some extent. - Highlights: • Cadmium could affect the expression levels of circadian rhythm-related genes. • Genes expression in microarray data were consistent with those in RT-PCR analysis. • Immune response and G protein-coupled receptor signaling pathway were identified. • Cadmium induces circadian rhythm disruption by different mechanism in day and night.« less

Diabetic cardiomyopathy is associated with defective myocellular copper regulation and both defects are rectified by divalent copper chelation

PubMed Central

2014-01-01

Background Heart disease is the leading cause of death in diabetic patients, and defective copper metabolism may play important roles in the pathogenesis of diabetic cardiomyopathy (DCM). The present study sought to determine how myocardial copper status and key copper-proteins might become impaired by diabetes, and how they respond to treatment with the Cu (II)-selective chelator triethylenetetramine (TETA) in DCM. Methods Experiments were performed in Wistar rats with streptozotocin (STZ)-induced diabetes with or without TETA treatment. Cardiac function was analyzed in isolated-perfused working hearts, and myocardial total copper content measured by particle-induced x-ray emission spectroscopy (PIXE) coupled with Rutherford backscattering spectrometry (RBS). Quantitative expression (mRNA and protein) and/or activity of key proteins that mediate LV-tissue-copper binding and transport, were analyzed by combined RT-qPCR, western blotting, immunofluorescence microscopy, and enzyme activity assays. Statistical analysis was performed using Student’s t-tests or ANOVA and p-values of < 0.05 have been considered significant. Results Left-ventricular (LV) copper levels and function were severely depressed in rats following 16-weeks’ diabetes, but both were unexpectedly normalized 8-weeks after treatment with TETA was instituted. Localized myocardial copper deficiency was accompanied by decreased expression and increased polymerization of the copper-responsive transition-metal-binding metallothionein proteins (MT1/MT2), consistent with impaired anti-oxidant defences and elevated susceptibility to pro-oxidant stress. Levels of the high-affinity copper transporter-1 (CTR1) were depressed in diabetes, consistent with impaired membrane copper uptake, and were not modified by TETA which, contrastingly, renormalized myocardial copper and increased levels and cell-membrane localization of the low-affinity copper transporter-2 (CTR2). Diabetes also lowered indexes of intracellular (IC) copper delivery via the copper chaperone for superoxide dismutase (CCS) to its target cuproenzyme, superoxide dismutase-1 (SOD1): this pathway was rectified by TETA treatment, which normalized SOD1 activity with consequent bolstering of anti-oxidant defenses. Furthermore, diabetes depressed levels of additional intracellular copper-transporting proteins, including antioxidant-protein-1 (ATOX1) and copper-transporting-ATPase-2 (ATP7B), whereas TETA elevated copper-transporting-ATPase-1 (ATP7A). Conclusions Myocardial copper deficiency and defective cellular copper transport/trafficking are revealed as key molecular defects underlying LV impairment in diabetes, and TETA-mediated restoration of copper regulation provides a potential new class of therapeutic molecules for DCM. PMID:24927960

Quantitative proteomic analysis of extracellular matrix extracted from mono- and dual-species biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis.

PubMed

Mohammed, Marwan Mansoor Ali; Pettersen, Veronika Kuchařová; Nerland, Audun H; Wiker, Harald G; Bakken, Vidar

2017-04-01

The Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis are members of a complex dental biofilm associated with periodontal disease. In this study, we cultured F. nucleatum and P. gingivalis as mono- and dual-species biofilms, and analyzed the protein composition of the biofilms extracellular polymeric matrix (EPM) by high-resolution liquid chromatography-tandem mass spectrometry. Label-free quantitative proteomic analysis was used for identification of proteins and sequence-based functional characterization for their classification and prediction of possible roles in EPM. We identified 542, 93 and 280 proteins in the matrix of F. nucleatum, P. gingivalis, and the dual-species biofilm, respectively. Nearly 70% of all EPM proteins in the dual-species biofilm originated from F. nucleatum, and a majority of these were cytoplasmic proteins, suggesting an enhanced lysis of F. nucleatum cells. The proteomic analysis also indicated an interaction between the two species: 22 F. nucleatum proteins showed differential levels between the mono and dual-species EPMs, and 11 proteins (8 and 3 from F. nucleatum and P. gingivalis, respectively) were exclusively detected in the dual-species EPM. Oxidoreductases and chaperones were among the most abundant proteins identified in all three EPMs. The biofilm matrices in addition contained several known and hypothetical virulence proteins, which can mediate adhesion to the host cells and disintegration of the periodontal tissues. This study demonstrated that the biofilm matrix of two important periodontal pathogens consists of a multitude of proteins whose amounts and functionalities vary largely. Relatively high levels of several of the detected proteins might facilitate their potential use as targets for the inhibition of biofilm development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteomic investigation into betulinic acid-induced apoptosis of human cervical cancer HeLa cells.

PubMed

Xu, Tao; Pang, Qiuying; Zhou, Dong; Zhang, Aiqin; Luo, Shaman; Wang, Yang; Yan, Xiufeng

2014-01-01

Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes 14-3-3β and 14-3-3ε. Furthermore, it was found that the antiapoptotic bcl-2 gene was down-regulated while the proapoptotic bax gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.

Exogenous DKK-3/REIC inhibits Wnt/β-catenin signaling and cell proliferation in human kidney cancer KPK1.

PubMed

Xu, Jiaqi; Sadahira, Takuya; Kinoshita, Rie; Li, Shun-Ai; Huang, Peng; Wada, Koichiro; Araki, Motoo; Ochiai, Kazuhiko; Noguchi, Hirofumi; Sakaguchi, Masakiyo; Nasu, Yasutomo; Watanabe, Masami

2017-11-01

The third member of the Dickkopf family (DKK-3), also known as reduced expression in immortalized cells (REIC), is a tumor suppressor present in a variety of tumor cells. Regarding the regulation of the Wnt/β-catenin signaling pathway, exogenous DKK-1 and DKK-2 are reported to inhibit Wnt signaling by binding the associated effectors. However, whether exogenous DKK-3 inhibits Wnt signaling remains unclear. A recombinant protein of human full-length DKK-3 was used to investigate the exogenous effects of the protein in vitro in KPK1 human renal cell carcinoma cells. It was demonstrated that the expression of phosphorylated (p-)β-catenin (inactive form as the transcriptional factor) was increased in KPK1 cells treated with the exogenous DKK-3 protein. The levels of non-p-β-catenin (activated form of β-catenin) were consistently decreased. It was revealed that the expression of transcription factor (TCF) 1 and c-Myc, the downstream transcription factors of the Wnt/β-catenin signaling pathway, was inhibited following treatment with DKK-3. A cancer cell viability assay confirmed the anti-proliferative effects of exogenous DKK-3 protein, which was consistent with a suppressed Wnt/β-catenin signaling cascade. In addition, as low-density lipoprotein receptor-related protein 6 (LRP6) is a receptor of DKK-1 and DKK-2 and their interaction on the cell surface inhibits Wnt/β-catenin signaling, it was examined whether the exogenous DKK-3 protein affects LRP6-mediated Wnt/β-catenin signaling. The LRP6 gene was silenced and the effects of DKK-3 on the time course of the upregulation of p-β-catenin expression were subsequently analyzed. Notably, LRP6 depletion elevated the base level of p-β-catenin; however, there was no significant effect on its upregulation course or expression pattern. These findings indicate that exogenous DKK-3 upregulates p-β-catenin and inhibits Wnt/β-catenin signaling in an LRP6-independent manner. Therefore, exogenous DKK-3 protein may inhibit the proliferation of KPK1 cells via inactivating Wnt/β-catenin signaling.

Effects of replacing fishmeal with animal by-products meal supplementation in diets on the growth and nutrient utilization of mangrove red snapper

NASA Astrophysics Data System (ADS)

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

2007-07-01

A feeding trial was conducted for 75 d to evaluate the nutritive value of a mixture of animal by-products (MAB) as a possible protein source in diets for juvenile mangrove red snapper, Lutjanus argentimaculatus (mean initial body weight, 30 g). Fish were fed one of five isonitrogenous diets (40% crude protein) replacing 0, 25% (MAB25), 50% (MAB50), 75% (MAB75) and 100% (MAB100) of fish meal protein with similar percentages of MAB. The MAB consisted of 25% cow liver meal, 20% leather meal, 20% meat and bone meal, 15% blood meal, 10% APC (poultry feather meal), 8% poultry manure dried, 1.5% choline and 0.5% chromic oxide. After 75 d of feeding, fish fed with diets MAB50, MAB75 and MAB100 exhibited significantly lower growth performance than that of fish fed with control and MAB25 diets. The optimum level of MAB was estimated to be 23%. Replacement of fish meal by MAB23% showed the following performance: maximum weight gain, 510%; SGR, 2.39% and FCE, 2.83%. The MAB substitution up to 75% of fish meal protein in diets did not show differences in apparent protein digestibility (83.6% for MAB25, 79.2% for MAB50, 78.7% for MAB75) compared with control (83.4%), whereas in MAB100 group digestibility (65.3%) was significantly lower than in other groups. The apparent phosphorus absorption of test diet groups was significantly higher (37.1% for MAB25, 28.5% for MAB50, 55.6% for MAB75 and 54.5% for MAB100) than that of control (11.2%). The levels of protein and ash in the whole body, carcass and viscera increased as MAB substitution in diets increased, whereas lipids and moisture remained consistent among all treatment groups. These results showed that approximately 23% of fish meal protein could be replaced by a mixture of animal by-products for juvenile snapper growing from 30 g to 167 g in 75 d without compromising growth performance and feed efficiency.

Dietary practices in propionic acidemia: A European survey.

PubMed

Daly, A; Pinto, A; Evans, S; Almeida, M F; Assoun, M; Belanger-Quintana, A; Bernabei, S M; Bollhalder, S; Cassiman, D; Champion, H; Chan, H; Dalmau, J; de Boer, F; de Laet, C; de Meyer, A; Desloovere, A; Dianin, A; Dixon, M; Dokoupil, K; Dubois, S; Eyskens, F; Faria, A; Fasan, I; Favre, E; Feillet, F; Fekete, A; Gallo, G; Gingell, C; Gribben, J; Kaalund Hansen, K; Ter Horst, N M; Jankowski, C; Janssen-Regelink, R; Jones, I; Jouault, C; Kahrs, G E; Kok, I L; Kowalik, A; Laguerre, C; Le Verge, S; Lilje, R; Maddalon, C; Mayr, D; Meyer, U; Micciche, A; Och, U; Robert, M; Rocha, J C; Rogozinski, H; Rohde, C; Ross, K; Saruggia, I; Schlune, A; Singleton, K; Sjoqvist, E; Skeath, R; Stolen, L H; Terry, A; Timmer, C; Tomlinson, L; Tooke, A; Vande Kerckhove, K; van Dam, E; van den Hurk, T; van der Ploeg, L; van Driessche, M; van Rijn, M; van Wegberg, A; Vasconcelos, C; Vestergaard, H; Vitoria, I; Webster, D; White, F J; White, L; Zweers, H; MacDonald, A

2017-12-01

The definitive dietary management of propionic acidaemia (PA) is unknown although natural protein restriction with adequate energy provision is of key importance. To describe European dietary practices in the management of patients with PA prior to the publication of the European PA guidelines. This was a cross-sectional survey consisting of 27 questions about the dietary practices in PA patients circulated to European IMD dietitians and health professionals in 2014. Information on protein restricted diets of 186 PA patients from 47 centres, representing 14 European countries was collected. Total protein intake [PA precursor-free L-amino acid supplements (PFAA) and natural protein] met WHO/FAO/UNU (2007) safe protein requirements for age in 36 centres (77%). PFAA were used to supplement natural protein intake in 81% (n = 38) of centres, providing a median of 44% (14-83%) of total protein requirement. Seventy-four per cent of patients were prescribed natural protein intakes below WHO/FAO/UNU (2007) safe levels in one or more of the following age groups: 0-6 m, 7-12 m, 1-10 y, 11-16 y and > 16 y. Sixty-three per cent (n = 117) of patients were tube fed (74% gastrostomy), but only 22% received nocturnal feeds. There was high use of PFAA with intakes of natural protein commonly below WHO/FAO/UNU (2007) safe levels. Optimal dietary management can only be determined by longitudinal, multi-centre, prospective case controlled studies. The metabolic instability of PA and small patient cohorts in each centre ensure that this is a challenging undertaking.

Cadmium inhibits the protein degradation of Sml1 by inhibiting the phosphorylation of Sml1 in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baek, In-Joon; Kang, Hyun-Jun; Chang, Miwha

Highlights: Black-Right-Pointing-Pointer Cd inhibits Sml1-p formation. Black-Right-Pointing-Pointer Cd affects cell cycle. Black-Right-Pointing-Pointer Cd inhibits Sml1 ubiquitination. -- Abstract: Cadmium is a toxic metal, and the mechanism of cadmium toxicity in living organisms has been well studied. Here, we used Saccharomyces cerevisiae as a model system to examine the detailed molecular mechanism of cell growth defects caused by cadmium. Using a plate assay of a yeast deletion mutant collection, we found that deletion of SML1, which encodes an inhibitor of Rnr1, resulted in cadmium resistance. Sml1 protein levels increased when cells were treated with cadmium, even though the mRNA levels ofmore » SML1 remained unchanged. Using northern and western blot analyses, we found that cadmium inhibited Sml1 degradation by inhibiting Sml1 phosphorylation. Sml1 protein levels increased when cells were treated with cadmium due to disruption of the dependent protein degradation pathway. Furthermore, cadmium promoted cell cycle progression into the G2 phase. The same result was obtained using cells in which SML1 was overexpressed. Deletion of SML1 delayed cell cycle progression. These results are consistent with Sml1 accumulation and with growth defects caused by cadmium stress. Interestingly, although cadmium treatment led to increase Sml1 levels, intracellular dNTP levels also increased because of Rnr3 upregulation due to cadmium stress. Taken together, these results suggest that cadmium specifically affects the phosphorylation of Sml1 and that Sml1 accumulates in cells.« less

Effect of a precompetition bodybuilding diet and training regimen on body composition and blood chemistry.

PubMed

Too, D; Wakayama, E J; Locati, L L; Landwer, G E

1998-09-01

The purpose of this investigation was to document the effect of a 10-wk precompetition bodybuilding diet and training, on blood chemistry and body composition. One adult male, steroid and drug free, preparing for a first competition. Average daily dietary intake consisted of 2263 calories (71% protein, 16% carbohydrate, 13% fats), with a protein intake of 5.0 gm.kg-1 body mass (BM). Initial body weight of 76.3 kgf (16% body fat) decreased to 63.4 kgf (4.4% body fat). Blood samples for electrolytes, TP, Alb, bilirubin, LDL-C, TG, UA, and amylase were normal. HDL-C levels increased from 65 to 89 mg.dL-1. Decreased glucose levels (< 50 mg.dL-1), indicated hypoglycemia. Increased Mg, LD, and CK levels indicated intense training. Increased inorganic phosphorus from 3.7 to 8.2 mg.dL-1 suggested lactic acidosis. Increased BUN levels from 16 to 53 mg.dL-1 and creatinine from 1.1 to 1.8 mg.dL-1 may be attributed to a high protein diet. However, heart muscle enzyme (CK-MB) was not elevated. Substantial changes in body composition and blood chemistry suggest adequate nutrition be ensured, and caution taken to avoid excessive physiologic stresses on the body during precompetition diet and training.

Single prolonged stress enhances hippocampal glucocorticoid receptor and phosphorylated protein kinase B levels

PubMed Central

Eagle, Andrew L.; Knox, Dayan; Roberts, Megan M.; Mulo, Kostika; Liberzon, Israel; Galloway, Matthew P.; Perrine, Shane A.

2012-01-01

Animal models of posttraumatic stress disorder (PTSD) can explore neurobiological mechanisms by which trauma enhances fear and anxiety reactivity. Single prolonged stress (SPS) shows good validity in producing PTSD-like behavior. While SPS-induced behaviors have been linked to enhanced glucocorticoid receptor (GR) expression, the molecular ramifications of enhanced GR expression have yet to be identified. Phosphorylated protein kinase B (pAkt) is critical for stress-mediated enhancement in general anxiety and memory, and may be regulated by GRs. However, it is currently unknown if pAkt levels are modulated by SPS, as well as if the specificity of GR and pAkt related changes contribute to anxiety-like behavior after SPS. The current study set out to examine the effects of SPS on GR and pAkt protein levels in the amygdala and hippocampus and to examine the specificity of these changes to unconditioned anxiety-like behavior. Levels of GR and pAkt were increased in the hippocampus, but not amygdala. Furthermore, SPS had no effect on unconditioned anxiety-like behavior suggesting that generalized anxiety is not consistently observed following SPS. The results suggest that SPS-enhanced GR expression is associated with phosphorylation of Akt, and also suggest that these changes are not related to an anxiogenic phenotype. PMID:23201176

The effect of preseason training on mucosal immunity in male basketball players.

PubMed

Azarbayjani, M; Nikbakht, H; Rasaee, M J

2011-12-01

This study examined the effects of pre season training on restring level and acute response of mucosal immunity in male basketball players. Twenty male basketball players performed 8 weeks progressive exercise training, consisting of interval and continuous parts. Five mL un-stimulated saliva was collected from each subject before, immediately and one hour after the end of one bout of exercise to exhaustion on treadmill at the beginning of the first week and end of 8 weeks to determine the acute responses. At the beginning of each 2 weeks (resting state) induced changes in basal mucosal immunity was evaluated. The concentration of sIgA and total protein was measured by the ELISA and Bradford methods respectively. One bout exercise training at beginning of first week decreased significantly sIgA level but not at the end of 8th week. Total protein did not change significantly at 1st week after exercise, but at eight week significantly increased and remained at high level until one hour after exercise. sIgA to total protein ratio at first week significantly decreased and remained constant one hour after exercise. At the eight week sIgA decreased significantly immediately after exercise and remained low until one hour after exercise. The comparison of sIgA and total protein levels indicates significant decrease after eight weeks training. These results suggest that repetition of single bout of exercise training have a cumulative effect on the mucosal immune system.

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells.

PubMed

Chen, Lisa S; Redkar, Sanjeev; Bearss, David; Wierda, William G; Gandhi, Varsha

2009-11-05

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-X(L), XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction.

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells

PubMed Central

Chen, Lisa S.; Redkar, Sanjeev; Bearss, David; Wierda, William G.

2009-01-01

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-XL, XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction. PMID:19734450

Comparative study of the efficacy of pulsed electromagnetic field and low level laser therapy on mitogen-activated protein kinases.

PubMed

El-Makakey, Ayman M; El-Sharaby, Radwa M; Hassan, Mohammed H; Balbaa, Alaa

2017-03-01

Mitogen-Activated Protein Kinases (MAPKs) consist of three major signaling members: extracellular signal-regulated kinase (ERK), p38 and C-JUN N-terminal kinase (JNK). We investigated physiological effects of Pulsed Electromagnetic Field Therapy (PEMFT) and Low Level Laser Therapy (LLLT) on human body, adopting the expression level of mitogen-activated protein kinases as an indicator via assessment of the activation levels of three major families of MAPKS, ERK, p38 and JNK in the peripheral lymphocytes of patients before and after the therapies. Assessment for the expression levels of MAPKs families' were done, in the peripheral lymphocytes of patients recently have appendectomy, using flow cytometric analysis of multiple signaling pathways, pre and post LLLT and PEMFT application (twice daily for 6 successive days) on the appendectomy wound. There were non-significant differences in the expression levels of MAPKs families' pre- therapies application. But there were significant increase in the ERK expression levels post application of LLLT compared to its pre application (p<0.01). Also, there was significant increase in the ERK, p38 and C-Jun N terminal expression level values post application of PEMFT compared to its pre application expression levels (p<0.01 for each). The present study demonstrates that PEMFT has a powerful healing effect more than LLLT as it increase the activation of ERK, P38 and C-Jun-N Terminal while LLLT only increase the activation of ERK. LLLT has more potent pain decreasing effect than PEMFT as it does not activate P38 pathway like PEMFT.

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells

PubMed Central

Singh, Satyendra; Klarmann, Kimberly D.; Coppola, Vincenzo; Keller, Jonathan R.; Tessarollo, Lino

2016-01-01

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies. PMID:27835883

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells.

PubMed

Puverel, Sandrine; Kiris, Erkan; Singh, Satyendra; Klarmann, Kimberly D; Coppola, Vincenzo; Keller, Jonathan R; Tessarollo, Lino

2016-12-20

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies.

Synthetic Peptide Arrays for Pathway-Level Protein Monitoring by Liquid Chromatography-Tandem Mass Spectrometry*

PubMed Central

Hewel, Johannes A.; Liu, Jian; Onishi, Kento; Fong, Vincent; Chandran, Shamanta; Olsen, Jonathan B.; Pogoutse, Oxana; Schutkowski, Mike; Wenschuh, Holger; Winkler, Dirk F. H.; Eckler, Larry; Zandstra, Peter W.; Emili, Andrew

2010-01-01

Effective methods to detect and quantify functionally linked regulatory proteins in complex biological samples are essential for investigating mammalian signaling pathways. Traditional immunoassays depend on proprietary reagents that are difficult to generate and multiplex, whereas global proteomic profiling can be tedious and can miss low abundance proteins. Here, we report a target-driven liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy for selectively examining the levels of multiple low abundance components of signaling pathways which are refractory to standard shotgun screening procedures and hence appear limited in current MS/MS repositories. Our stepwise approach consists of: (i) synthesizing microscale peptide arrays, including heavy isotope-labeled internal standards, for use as high quality references to (ii) build empirically validated high density LC-MS/MS detection assays with a retention time scheduling system that can be used to (iii) identify and quantify endogenous low abundance protein targets in complex biological mixtures with high accuracy by correlation to a spectral database using new software tools. The method offers a flexible, rapid, and cost-effective means for routine proteomic exploration of biological systems including “label-free” quantification, while minimizing spurious interferences. As proof-of-concept, we have examined the abundance of transcription factors and protein kinases mediating pluripotency and self-renewal in embryonic stem cell populations. PMID:20467045

RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage

PubMed Central

Dai, Weijun; Zhang, Gen; Makeyev, Eugene V.

2012-01-01

RNA-binding protein HuR modulates the stability and translational efficiency of messenger RNAs (mRNAs) encoding essential components of the cellular proliferation, growth and survival pathways. Consistent with these functions, HuR levels are often elevated in cancer cells and reduced in senescent and quiescent cells. However, the molecular mechanisms that control HuR expression are poorly understood. Here we show that HuR protein autoregulates its abundance through a negative feedback loop that involves interaction of the nuclear HuR protein with a GU-rich element (GRE) overlapping with the HuR major polyadenylation signal (PAS2). An increase in the cellular HuR protein levels stimulates the expression of long HuR mRNA species containing an AU-rich element (ARE) that destabilizes the mRNAs and thus reduces the protein production output. The PAS2 read-through occurs due to a reduced recruitment of the CstF-64 subunit of the pre-mRNA cleavage stimulation factor in the presence of the GRE-bound HuR. We propose that this mechanism maintains HuR homeostasis in proliferating cells. Since only the nuclear HuR is expected to contribute to the auto-regulation, our model may explain the longstanding observation that the increase in the total HuR expression in cancer cells often correlates with the accumulation of its substantial fraction in the cytoplasm. PMID:21948791

RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage.

PubMed

Dai, Weijun; Zhang, Gen; Makeyev, Eugene V

2012-01-01

RNA-binding protein HuR modulates the stability and translational efficiency of messenger RNAs (mRNAs) encoding essential components of the cellular proliferation, growth and survival pathways. Consistent with these functions, HuR levels are often elevated in cancer cells and reduced in senescent and quiescent cells. However, the molecular mechanisms that control HuR expression are poorly understood. Here we show that HuR protein autoregulates its abundance through a negative feedback loop that involves interaction of the nuclear HuR protein with a GU-rich element (GRE) overlapping with the HuR major polyadenylation signal (PAS2). An increase in the cellular HuR protein levels stimulates the expression of long HuR mRNA species containing an AU-rich element (ARE) that destabilizes the mRNAs and thus reduces the protein production output. The PAS2 read-through occurs due to a reduced recruitment of the CstF-64 subunit of the pre-mRNA cleavage stimulation factor in the presence of the GRE-bound HuR. We propose that this mechanism maintains HuR homeostasis in proliferating cells. Since only the nuclear HuR is expected to contribute to the auto-regulation, our model may explain the longstanding observation that the increase in the total HuR expression in cancer cells often correlates with the accumulation of its substantial fraction in the cytoplasm.

Systems-wide analysis of manganese deficiency-induced changes in gene activity of Arabidopsis roots

PubMed Central

Rodríguez-Celma, Jorge; Tsai, Yi-Hsiu; Wen, Tuan-Nan; Wu, Yu-Ching; Curie, Catherine; Schmidt, Wolfgang

2016-01-01

Manganese (Mn) is pivotal for plant growth and development, but little information is available regarding the strategies that evolved to improve Mn acquisition and cellular homeostasis of Mn. Using an integrated RNA-based transcriptomic and high-throughput shotgun proteomics approach, we generated a comprehensive inventory of transcripts and proteins that showed altered abundance in response to Mn deficiency in roots of the model plant Arabidopsis. A suite of 22,385 transcripts was consistently detected in three RNA-seq runs; LC-MS/MS-based iTRAQ proteomics allowed the unambiguous determination of 11,606 proteins. While high concordance between mRNA and protein expression (R = 0.87) was observed for transcript/protein pairs in which both gene products accumulated differentially upon Mn deficiency, only approximately 10% of the total alterations in the abundance of proteins could be attributed to transcription, indicating a large impact of protein-level regulation. Differentially expressed genes spanned a wide range of biological functions, including the maturation, translation, and transport of mRNAs, as well as primary and secondary metabolic processes. Metabolic analysis by UPLC-qTOF-MS revealed that the steady-state levels of several major glucosinolates were significantly altered upon Mn deficiency in both roots and leaves, possibly as a compensation for increased pathogen susceptibility under conditions of Mn deficiency. PMID:27804982

Botulinum neurotoxin: a marvel of protein design.

PubMed

Montal, Mauricio

2010-01-01

Botulinum neurotoxin (BoNT), the causative agent of botulism, is acknowledged to be the most poisonous protein known. BoNT proteases disable synaptic vesicle exocytosis by cleaving their cytosolic SNARE (soluble NSF attachment protein receptor) substrates. BoNT is a modular nanomachine: an N-terminal Zn(2+)-metalloprotease, which cleaves the SNAREs; a central helical protein-conducting channel, which chaperones the protease across endosomes; and a C-terminal receptor-binding module, consisting of two subdomains that determine target specificity by binding to a ganglioside and a protein receptor on the cell surface and triggering endocytosis. For BoNT, functional complexity emerges from its modular design and the tight interplay between its component modules--a partnership with consequences that surpass the simple sum of the individual component's action. BoNTs exploit this design at each step of the intoxication process, thereby achieving an exquisite toxicity. This review summarizes current knowledge on the structure of individual modules and presents mechanistic insights into how this protein machine evolved to this level of sophistication. Understanding the design principles underpinning the function of such a dynamic modular protein remains a challenging task.

Large scale systematic proteomic quantification from non-metastatic to metastatic colorectal cancer

NASA Astrophysics Data System (ADS)

Yin, Xuefei; Zhang, Yang; Guo, Shaowen; Jin, Hong; Wang, Wenhai; Yang, Pengyuan

2015-07-01

A systematic proteomic quantification of formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tissues from stage I to stage IIIC was performed in large scale. 1017 proteins were identified with 338 proteins in quantitative changes by label free method, while 341 proteins were quantified with significant expression changes among 6294 proteins by iTRAQ method. We found that proteins related to migration expression increased and those for binding and adherent decreased during the colorectal cancer development according to the gene ontology (GO) annotation and ingenuity pathway analysis (IPA). The integrin alpha 5 (ITA5) in integrin family was focused, which was consistent with the metastasis related pathway. The expression level of ITA5 decreased in metastasis tissues and the result has been further verified by Western blotting. Another two cell migration related proteins vitronectin (VTN) and actin-related protein (ARP3) were also proved to be up-regulated by both mass spectrometry (MS) based quantification results and Western blotting. Up to now, our result shows one of the largest dataset in colorectal cancer proteomics research. Our strategy reveals a disease driven omics-pattern for the metastasis colorectal cancer.

Dietary protein for athletes: from requirements to metabolic advantage.

PubMed

Phillips, Stuart M

2006-12-01

The Dietary Reference Intakes (DRI) specify that the requirement for dietary protein for all individuals aged 19 y and older is 0.8 g protein.kg-1.d-1. This Recommended Dietary Allowance (RDA) is cited as adequate for all persons. This amount of protein would be considered by many athletes as the amount to be consumed in a single meal, particularly for strength-training athletes. There does exist, however, published data to suggest that individuals habitually performing resistance and (or) endurance exercise require more protein than their sedentary counterparts. The RDA values for protein are clearly set at "...the level of protein judged to be adequate... to meet the known nutrient needs for practically all healthy people...". The RDA covers protein losses with margins for inter-individual variability and protein quality; the notion of consumption of excess protein above these levels to cover increased needs owing to physical activity is not, however, given any credence. Notwithstanding, diet programs (i.e., energy restriction) espousing the virtue of high protein enjoy continued popularity. A number of well-controlled studies are now published in which "higher" protein diets have been shown to be effective in promoting weight reduction, particularly fat loss. The term "higher" refers to a diet that has people consuming more than the general populations' average intake of approximately 15% of energy from protein, e.g., as much as 30%-35%, which is within an Acceptable Macronutrient Distribution Range (AMDR) as laid out in the DRIs. Of relevance to athletes and those in clinical practice is the fact that higher protein diets have quite consistently been shown to result in greater weight loss, greater fat loss, and preservation of lean mass as compared with "lower" protein diets. A framework for understanding dietary protein intake within the context of weight loss and athletic performance is laid out.

Comparison of the pharmacological profiles of murine antisense oligonucleotides targeting apolipoprotein B and microsomal triglyceride transfer protein

PubMed Central

Lee, Richard G.; Fu, Wuxia; Graham, Mark J.; Mullick, Adam E.; Sipe, Donna; Gattis, Danielle; Bell, Thomas A.; Booten, Sheri; Crooke, Rosanne M.

2013-01-01

Therapeutic agents that suppress apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) levels/activity are being developed in the clinic to benefit patients who are unable to reach target LDL-C levels with maximally tolerated lipid-lowering drugs. To compare and contrast the metabolic consequences of reducing these targets, murine-specific apoB or MTP antisense oligonucleotides (ASOs) were administered to chow-fed and high fat-fed C57BL/6 or to chow-fed and Western diet-fed LDLr−/− mice for periods ranging from 2 to 12 weeks, and detailed analyses of various factors affecting fatty acid metabolism were performed. Administration of these drugs significantly reduced target hepatic mRNA and protein, leading to similar reductions in hepatic VLDL/triglyceride secretion. MTP ASO treatment consistently led to increases in hepatic triglyceride accumulation and biomarkers of hepatotoxicity relative to apoB ASO due in part to enhanced expression of peroxisome proliferator activated receptor γ target genes and the inability to reduce hepatic fatty acid synthesis. Thus, although both drugs effectively lowered LDL-C levels in mice, the apoB ASO produced a more positive liver safety profile. PMID:23220583

Expression and Activity of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Distal Lung Epithelial Cells In Vitro.

PubMed

Nickel, Sabrina; Selo, Mohammed Ali; Fallack, Juliane; Clerkin, Caoimhe G; Huwer, Hanno; Schneider-Daum, Nicole; Lehr, Claus-Michael; Ehrhardt, Carsten

2017-12-01

Breast cancer resistance protein (BCRP/ABCG2) has previously been identified with high expression levels in human lung. The subcellular localisation and functional activity of the transporter in lung epithelia, however, remains poorly investigated. The aim of this project was to study BCRP expression and activity in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and to compare these findings with data obtained from the NCI-H441 cell line. BCRP expression levels in AT2 and AT1-like cells and in different passages of NCI-H441 cells were determined using q-PCR and immunoblot. Transporter localisation was confirmed by confocal laser scanning microscopy. Efflux and transport studies using the BCRP substrate BODIPY FL prazosin and the inhibitor Ko143 were carried out to assess BCRP activity in the different cell models. BCRP expression decreased during transdifferentiation from AT2 to AT1-like phenotype. Culturing NCI-H441 cells at an air-liquid interface or submersed did not change BCRP abundance, however, BCRP levels increased with passage number. BCRP was localised to the apical membrane and cytosol in NCI-H441 cells. In primary cells, the protein was found predominantly in the nucleus. Functional studies were consistent with expression data. BCRP is differently expressed in AT2 and AT1-like cells with lower abundance and activity in the latter ones. Nuclear BCRP might play a transcriptional role in distal lung epithelium. In NCI-H441 cells, BCRP is expressed in apical cell membranes and its activity is consistent with the localisation pattern.

Effects of protein or amino-acid supplementation on the physical growth of young children in low-income countries

PubMed Central

Arsenault, Joanne E; Brown, Kenneth H

2017-01-01

Abstract Child growth stunting is common in low-income countries, possibly due to insufficient protein intakes. Most previous studies have concluded that children’s protein intakes are adequate in relation to estimated requirements, but these studies did not consider issues of protein digestibility and effects of infection on dietary protein utilization. Using an alternative approach to assess the possible role of protein inadequacy in children’s growth restriction, the results of 18 intervention trials in which supplementary protein or amino acids were provided to children ages 6–35 months and growth outcomes were reviewed. Eight studies conducted in hospitalized children recovering from acute malnutrition found that the recommended protein intake levels for healthy children supported normal growth rates, but higher intakes were needed for accelerated rates of “catch-up” growth. Ten community-based studies did not demonstrate a consistent benefit of supplemental protein on children’s growth. However, weaknesses in the study designs limit the conclusions that can be drawn from these studies, and additional appropriately designed trials are needed to answer this question definitively. Recommendations for optimizing future study designs are provided herein. PMID:28938793

Dietary purines in vegetarian meat analogues.

PubMed

Havlik, Jaroslav; Plachy, Vladimir; Fernandez, Javier; Rada, Vojtech

2010-11-01

The meat alternatives market offers a wide range of products resembling meat in taste, flavour or texture but based on vegetable protein sources. These high protein-low purine foods may find application in a low purine or purine-free diet, which is sometimes suggested for subjects with increased serum urate levels, i.e. hyperuricaemia. We determined purine content (uric acid, adenine, guanine, hypoxanthine, xanthine) in 39 commercially available meat substitutes and evaluated them in relation to their protein content. Some of the products contained a comparable sum of adenine and hypoxanthine per protein as meat. Analysis of variance showed an influence of protein source used. Mycoprotein-based products had significantly higher contents (2264 mg kg(-1)) of adenine and hypoxanthine per kg of 100% protein than soybean-based products (1648 mg kg(-1)) or mixtures consisting of soybean protein and wheat protein (1239 mg kg(-1)). Protein-rich vegetable-based meat substitutes might be generally accepted as meat alternatives for individuals on special diets. The type of protein used to manufacture these products determines the total content of purines, which is relatively higher in the case of mycoprotein or soybean protein, while appearing lower in wheat protein and egg white-based products. These are therefore more suitable for dietary considerations in a low-purine diet for hyperuricaemic subjects. 2010 Society of Chemical Industry

Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells*

PubMed Central

de Queiroz, Rafaela Muniz; Madan, Rashna; Chien, Jeremy; Dias, Wagner Barbosa; Slawson, Chad

2016-01-01

O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked β-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function. Because ovarian cancer has a high frequency of p53 mutation rates, we decided to investigate the relationship between O-GlcNAcylation and p53 function in ovarian cancer. We measured a significant decrease in O-GlcNAcylation of tumor tissue in an ovarian tumor microarray. Furthermore, O-GlcNAcylation was increased, and OGA protein and mRNA levels were decreased in ovarian tumor cell lines not expressing the protein p53. Treatment with the OGA inhibitor Thiamet-G (TMG), silencing of OGA, or overexpression of OGA and OGT led to p53 stabilization, increased nuclear localization, and increased protein and mRNA levels of p53 target genes. These data suggest that changes in O-GlcNAc homeostasis activate the p53 pathway. Combination treatment of the chemotherapeutic cisplatin with TMG decreased tumor cell growth and enhanced cell cycle arrest without impairing cytotoxicity. The effects of TMG on tumor cell growth were partially dependent on wild type p53 activation. In conclusion, changes in O-GlcNAc homeostasis activate the wild type p53 pathway in ovarian cancer cells, and OGA inhibition has the potential as an adjuvant treatment for ovarian carcinoma. PMID:27402830

A Practical Standardized Composite Nutrition Score Based on Lean Tissue Index: Application in Nutrition Screening and Prediction of Outcome in Hemodialysis Population.

PubMed

Chen, Huan-Sheng; Cheng, Chun-Ting; Hou, Chun-Cheng; Liou, Hung-Hsiang; Chang, Cheng-Tsung; Lin, Chun-Ju; Wu, Tsai-Kun; Chen, Chang-Hsu; Lim, Paik-Seong

2017-07-01

Rapid screening and monitoring of nutritional status is mandatory in hemodialysis population because of the increasingly encountered nutritional problems. Considering the limitations of previous composite nutrition scores applied in this population, we tried to develop a standardized composite nutrition score (SCNS) using low lean tissue index as a marker of protein wasting to facilitate clinical screening and monitoring and to predict outcome. This retrospective cohort used 2 databases of dialysis populations from Taiwan between 2011 and 2014. First database consisting of data from 629 maintenance hemodialysis patients was used to develop the SCNS and the second database containing data from 297 maintenance hemodialysis patients was used to validate this developed score. SCNS containing albumin, creatinine, potassium, and body mass index was developed from the first database using low lean tissue index as a marker of protein wasting. When applying this score in the original database, significantly higher risk of developing protein wasting was found for patients with lower SCNS (odds ratio 1.38 [middle tertile vs highest tertile, P < .0001] and 2.40 [lowest tertile vs middle tertile, P < .0001]). The risk of death was also shown to be higher for patients with lower SCNS (hazard ratio 4.45 [below median level vs above median level, P < .0001]). These results were validated in the second database. We developed an SCNS consisting of 4 easily available biochemical parameters. This kind of scoring system can be easily applied in different dialysis facilities for screening and monitoring of protein wasting. The wide application of body composition monitor in dialysis population will also facilitate the development of specific nutrition scoring model for individual facility. Copyright © 2017 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

Proteomic and Bioinformatic Studies for the Characterization of Response to Pemetrexed in Platinum Drug Resistant Ovarian Cancer.

PubMed

Severi, Leda; Losi, Lorena; Fonda, Sergio; Taddia, Laura; Gozzi, Gaia; Marverti, Gaetano; Magni, Fulvio; Chinello, Clizia; Stella, Martina; Sheouli, Jalid; Braicu, Elena I; Genovese, Filippo; Lauriola, Angela; Marraccini, Chiara; Gualandi, Alessandra; D'Arca, Domenico; Ferrari, Stefania; Costi, Maria P

2018-01-01

Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuest™ tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection.

Molecular characterization of cDNA encoding oxygen evolving enhancer protein 1 increased by salt treatment in the mangrove Bruguiera gymnorrhiza.

PubMed

Sugihara, K; Hanagata, N; Dubinsky, Z; Baba, S; Karube, I

2000-11-01

Young plants of the common Okinawa mangrove species Bruguiera gymnorrhiza were transferred from freshwater to a medium with seawater salt level (500 mM NaCl). Two-dimensional gel electrophoresis revealed in the leaf extract of the plant a 33 kDa protein with pI 5.2, whose quantity increased as a result of NaCl treatment. The N-terminal amino acids sequence of this protein had a significant homology with mature region of oxygen evolving enhancer protein 1 (OEE1) precursor. The cloning of OEE1 precursor cDNA fragment was carried out by means of reverse transcription-PCR (RT-PCR) using degenerated primers. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA ends (RACE) method. The deduced amino acid sequence consisted of 322 amino acids and was 87% identical to that of Nicotiana tabacum. In B. gymnorrhiza, the predicted amino acid sequence of the mature protein starts at the residue number 85 of the open reading frame. The first 84-amino acid residues correspond to a typical transit sequence for the signal directing OEE1 to its appropriate compartment of chloroplast. The expression of OEE1 was analyzed together with other OEE subunits and D1 protein of photosystem II. The transcript levels of all the three OEEs were enhanced by NaCl treatment, but the significant increase of D1 protein was not observed.

Proteomic analysis of common bean seed with storage protein deficiency reveals up-regulation of sulfur-rich proteins and starch and raffinose metabolic enzymes, and down-regulation of the secretory pathway.

PubMed

Marsolais, Frédéric; Pajak, Agnieszka; Yin, Fuqiang; Taylor, Meghan; Gabriel, Michelle; Merino, Diana M; Ma, Vanessa; Kameka, Alexander; Vijayan, Perumal; Pham, Hai; Huang, Shangzhi; Rivoal, Jean; Bett, Kirstin; Hernández-Sebastià, Cinta; Liu, Qiang; Bertrand, Annick; Chapman, Ralph

2010-06-16

A deficiency in major seed storage proteins is associated with a nearly two-fold increase in sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris). Their mature seed proteome was compared by an approach combining label-free quantification by spectral counting, 2-DE, and analysis of selective extracts. Lack of phaseolin, phytohemagglutinin and arcelin was mainly compensated by increases in legumin, alpha-amylase inhibitors and mannose lectin FRIL. Along with legumin, albumin-2, defensin and albumin-1 were major contributors to the elevated sulfur amino acid content. Coordinate induction of granule-bound starch synthase I, starch synthase II-2 and starch branching enzyme were associated with minor alteration of starch composition, whereas increased levels of UDP-glucose 4-epimerase were correlated with a 30% increase in raffinose content. Induction of cell division cycle protein 48 and ubiquitin suggested enhanced ER-associated degradation. This was not associated with a classical unfolded protein response as the levels of ER HSC70-cognate binding protein were actually reduced in the mutant. Repression of rab1 GTPase was consistent with decreased traffic through the secretory pathway. Collectively, these results have implications for the nutritional quality of common bean, and provide information on the pleiotropic phenotype associated with storage protein deficiency in a dicotyledonous seed. Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.

Application of wide selected-ion monitoring data-independent acquisition to identify tomato fruit proteins regulated by the CUTIN DEFICIENT2 transcription factor.

PubMed

Martin, Laetitia B B; Sherwood, Robert W; Nicklay, Joshua J; Yang, Yong; Muratore-Schroeder, Tara L; Anderson, Elizabeth T; Thannhauser, Theodore W; Rose, Jocelyn K C; Zhang, Sheng

2016-08-01

We describe here the use of label-free wide selected-ion monitoring data-independent acquisition (WiSIM-DIA) to identify proteins that are involved in the formation of tomato (Solanum lycopersicum) fruit cuticles and that are regulated by the transcription factor CUTIN DEFICIENT2 (CD2). A spectral library consisting of 11 753 unique peptides, corresponding to 2338 tomato protein groups, was used and the DIA analysis was performed at the MS1 level utilizing narrow mass windows for extraction with Skyline 2.6 software. We identified a total of 1140 proteins, 67 of which had expression levels that differed significantly between the cd2 tomato mutant and the wild-type cultivar M82. Differentially expressed proteins including a key protein involved in cutin biosynthesis, were selected for validation by target SRM/MRM and by Western blot analysis. In addition to confirming a role for CD2 in regulating cuticle formation, the results also revealed that CD2 influences pathways associated with cell wall biology, anthocyanin biosynthesis, plant development, and responses to stress, which complements findings of earlier RNA-Seq experiments. Our results provide new insights into molecular processes and aspects of fruit biology associated with CD2 function, and demonstrate that the WiSIM-DIA is an effective quantitative approach for global protein identifications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Autophagy and Its Impact on Neurodegenerative Diseases: New Roles for TDP-43 and C9orf72

PubMed Central

Budini, Mauricio; Buratti, Emanuele; Morselli, Eugenia; Criollo, Alfredo

2017-01-01

Autophagy is a catabolic mechanism where intracellular material is degraded by vesicular structures called autophagolysosomes. Autophagy is necessary to maintain the normal function of the central nervous system (CNS), avoiding the accumulation of misfolded and aggregated proteins. Consistently, impaired autophagy has been associated with the pathogenesis of various neurodegenerative diseases. The proteins TAR DNA-binding protein-43 (TDP-43), which regulates RNA processing at different levels, and chromosome 9 open reading frame 72 (C9orf72), probably involved in membrane trafficking, are crucial in the development of neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Additionally, recent studies have identified a role for these proteins in the control of autophagy. In this manuscript, we review what is known regarding the autophagic mechanism and discuss the involvement of TDP-43 and C9orf72 in autophagy and their impact on neurodegenerative diseases. PMID:28611593

Autophagy and Its Impact on Neurodegenerative Diseases: New Roles for TDP-43 and C9orf72.

PubMed

Budini, Mauricio; Buratti, Emanuele; Morselli, Eugenia; Criollo, Alfredo

2017-01-01

Autophagy is a catabolic mechanism where intracellular material is degraded by vesicular structures called autophagolysosomes. Autophagy is necessary to maintain the normal function of the central nervous system (CNS), avoiding the accumulation of misfolded and aggregated proteins. Consistently, impaired autophagy has been associated with the pathogenesis of various neurodegenerative diseases. The proteins TAR DNA-binding protein-43 (TDP-43), which regulates RNA processing at different levels, and chromosome 9 open reading frame 72 (C9orf72), probably involved in membrane trafficking, are crucial in the development of neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Additionally, recent studies have identified a role for these proteins in the control of autophagy. In this manuscript, we review what is known regarding the autophagic mechanism and discuss the involvement of TDP-43 and C9orf72 in autophagy and their impact on neurodegenerative diseases.

The PNPLA3 variant associated with fatty liver disease (I148M) accumulates on lipid droplets by evading ubiquitylation

PubMed Central

BasuRay, Soumik; Smagris, Eriks

2017-01-01

A sequence variation (I148M) in patatin‐like phospholipase domain‐containing protein 3 (PNPLA3) is strongly associated with fatty liver disease, but the underlying mechanism remains obscure. In this study, we used knock‐in (KI) mice (Pnpla3148M/M) to examine the mechanism responsible for accumulation of triglyceride (TG) and PNPLA3 in hepatic lipid droplets (LDs). No differences were found between Pnpla3148M/M and Pnpla3+/+ mice in hepatic TG synthesis, utilization, or secretion. These results are consistent with TG accumulation in the Pnpla3148M/M mice being caused by impaired TG mobilization from LDs. Sucrose feeding, which is required to elicit fatty liver in KI mice, led to a much larger and more persistent increase in PNPLA3 protein in the KI mice than in wild‐type (WT) mice. Inhibition of the proteasome (bortezomib), but not macroautophagy (3‐methyladenine), markedly increased PNPLA3 levels in WT mice, coincident with the appearance of ubiquitylated forms of the protein. Bortezomib did not increase PNPLA3 levels in Pnpla3148M/M mice, and only trace amounts of ubiquitylated PNPLA3 were seen in these animals. Conclusion: These results are consistent with the notion that the 148M variant disrupts ubiquitylation and proteasomal degradation of PNPLA3, resulting in accumulation of PNPLA3‐148M and impaired mobilization of TG from LDs. (Hepatology 2017;66:1111‐1124). PMID:28520213

Analysis on the expression and function of syndecan in the Pacific white shrimp Litopenaeus vannamei.

PubMed

Yang, Hui; Li, Shihao; Li, Fuhua; Wen, Rong; Xiang, Jianhai

2015-08-01

Syndecan is considered to be a multifunctional protein which functions as a cell surface receptor involved in cell adhesion, migration, cytoskeleton organization and differentiation. Previous bioinformatic analysis has revealed that syndecan in shrimp might interact with white spot syndrome virus (WSSV). In the present study, we experimentally studied the function of syndecan in shrimp immunity. The syndecan from Litopenaeus vannamei (LvSDC) was cloned and analyzed. The full-length cDNA of LvSDC was 1005 bp, consisting of 59 bp 5'-UTR, 253 bp 3'-UTR, and 693 bp open reading frame encoding 230 amino acids. LvSDC consisted of an extracellular domain (ED), a transmembrane domain (TM) and a cytoplasmic domain (CD). TM and CD shared high similarities with those of syndecan proteins from other species. LvSDC was ubiquitously expressed in all tested tissues, with the highest level in Oka. After WSSV challenge, the transcription level of LvSDC in Oka was apparently up-regulated. Recombinant LvSDC protein and its rabbit polyclonal antibody were prepared for detecting the location of LvSDC in hemocytes using immunocytochemistry approach. Data showed that LvSDC mainly located at the cell membrane and the cytoplasm of hemocytes. After silencing of LvSDC with siRNA, the WSSV copy numbers and mortality of shrimp after WSSV infection were both significantly decreased. These data provide useful information for understanding the immune mechanism of shrimp to WSSV infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chlorophyll a/b-binding proteins, pigment conversions, and early light-induced proteins in a chlorophyll b-less barley mutant.

PubMed Central

Król, M; Spangfort, M D; Huner, N P; Oquist, G; Gustafsson, P; Jansson, S

1995-01-01

Monospecific polyclonal antibodies have been raised against synthetic peptides derived from the primary sequences from different plant light-harvesting Chl a/b-binding (LHC) proteins. Together with other monospecific antibodies, these were used to quantify the levels of the 10 different LHC proteins in wild-type and chlorina f2 barley (Hordeum vulgare L.), grown under normal and intermittent light (ImL). Chlorina f2, grown under normal light, lacked Lhcb1 (type I LHC II) and Lhcb6 (CP24) and had reduced amounts of Lhcb2, Lhcb3 (types II and III LHC II), and Lhcb4 (CP 29). Chlorina f2 grown under ImL lacked all LHC proteins, whereas wild-type ImL plants contained Lhcb5 (CP 26) and a small amount of Lhcb2. The chlorina f2 ImL thylakoids were organized in large parallel arrays, but wild-type ImL thylakoids had appressed regions, indicating a possible role for Lhcb5 in grana stacking. Chlorina f2 grown under ImL contained considerable amounts of violaxanthin (2-3/reaction center), representing a pool of phototransformable xanthophyll cycle pigments not associated with LHC proteins. Chlorina f2 and the plants grown under ImL also contained early light-induced proteins (ELIPs) as monitored by western blotting. The levels of both ELIPs and xanthophyll cycle pigments increased during a 1 h of high light treatment, without accumulation of LHC proteins. These data are consistent with the hypothesis that ELIPs are pigment-binding proteins, and we suggest that ELIPs bind photoconvertible xanthophylls and replace "normal" LHC proteins under conditions of light stress. PMID:7748263

Diet Modeling in Older Americans: The Impact of Increasing Plant-Based Foods or Dairy Products on Protein Intake.

PubMed

Houchins, J A; Cifelli, C J; Demmer, E; Fulgoni, V L

2017-01-01

To determine the effects of increasing plant-based foods or dairy products on protein intake in older Americans by performing diet modeling. Data from What We Eat in America (WWEIA), the dietary component of the National Health and Nutrition Examination Survey (NHANES), 2007-2010 for Americans aged 51 years and older (n=5,389), divided as 51-70 years (n=3,513) and 71 years and older (n=1,876) were used. Usual protein intake was compared among three dietary models that increased intakes by 100%: (1) plant-based foods; (2) higher protein plant-based foods (i.e., legumes, nuts, seeds, soy); and (3) dairy products (milk, cheese, and yogurt). Models (1) and (2) had commensurate reductions in animal-based protein intake. Doubling intake of plant-based foods (as currently consumed) resulted in a drop of protein intake by approximately 22% for males and females aged 51+ years. For older males and females, aged 71+ years, doubling intake of plant-based foods (as currently consumed) resulted in an estimated usual intake of 0.83±0.02 g/kg ideal body weight (iBW))/day and 0.78±0.01 g/kg iBW/day, respectively. In this model, 33% of females aged 71+ years did not meet the estimated average requirement for protein. Doubling dairy product consumption achieved current protein intake recommendations. These data illustrate that increasing plant-based foods and reducing animal-based products could have unintended consequences on protein intake of older Americans. Doubling dairy product intake can help older adults get to an intake level of approximately 1.2 g/kg iBW/day, consistent with the growing consensus that older adults need to consume higher levels of protein for health.

Observation of giant conductance fluctuations in a protein

NASA Astrophysics Data System (ADS)

Zhang, Bintian; Song, Weisi; Pang, Pei; Zhao, Yanan; Zhang, Peiming; Csabai, István; Vattay, Gábor; Lindsay, Stuart

2017-12-01

Proteins are insulating molecular solids, yet even those containing easily reduced or oxidized centers can have single-molecule electronic conductances that are too large to account for with conventional transport theories. Here, we report the observation of remarkably high electronic conductance states in an electrochemically inactive protein, the ∼200 kD α V β 3 extracellular domain of human integrin. Large current pulses (up to nA) were observed for long durations (many ms, corresponding to many pC of charge transfer) at large gap (>5 nm) distances in an STM when the protein was bound specifically by a small peptide ligand attached to the electrodes. The effect is greatly reduced when a homologous, weakly binding protein (α 4 β 1) is used as a control. In order to overcome the limitations of the STM, the time- and voltage-dependence of the conductance were further explored using a fixed-gap (5 nm) tunneling junction device that was small enough to trap a single protein molecule at any one time. Transitions to a high conductance (∼nS) state were observed, the protein being ‘on’ for times from ms to tenths of a second. The high-conductance states only occur above ∼100 mV applied bias, and thus are not an equilibrium property of the protein. Nanoamp two-level signals indicate the specific capture of a single molecule in an electrode gap functionalized with the ligand. This offers a new approach to label-free electronic detection of single protein molecules. Electronic structure calculations yield a distribution of energy level spacings that is consistent with a recently proposed quantum-critical state for proteins, in which small fluctuations can drive transitions between localized and band-like electronic states.

Observation of Giant Conductance Fluctuations in a Protein

PubMed Central

Zhang, Bintian; Song, Weisi; Pang, Pei; Zhao, Yanan; Zhang, Peiming; Csabai, István; Vattay, Gábor; Lindsay, Stuart

2017-01-01

Proteins are insulating molecular solids, yet even those containing easily reduced or oxidized centers can have single-molecule electronic conductances that are too large to account for with conventional transport theories. Here, we report the observation of remarkably high electronic conductance states in an electrochemically-inactive protein, the ~200 kD αVβ3 extracelluar domain of human integrin. Large current pulses (up to nA) were observed for long durations (many ms, corresponding to many pC of charge transfer) at large gap (>5nm) distances in an STM when the protein was bound specifically by a small peptide ligand attached to the electrodes. The effect is greatly reduced when a homologous, weakly-binding protein (α4β1) is used as a control. In order to overcome the limitations of the STM, the time- and voltage-dependence of the conductance were further explored using a fixed-gap (5 nm) tunneling junction device that was small enough to trap a single protein molecule at any one time. Transitions to a high conductance (~ nS) state were observed, the protein being “on” for times from ms to tenths of a second. The high-conductance states only occur above ~ 100mV applied bias, and thus are not an equilibrium property of the protein. Nanoamp two-level signals indicate the specific capture of a single molecule in an electrode gap functionalized with the ligand. This offers a new approach to label-free electronic detection of single protein molecules. Electronic structure calculations yield a distribution of energy level spacings that is consistent with a recently proposed quantum-critical state for proteins, in which small fluctuations can drive transitions between localized and band-like electronic states. PMID:29552645

Effects of grain source, grain processing, and protein degradability on rumen kinetics and microbial protein synthesis in Boer kids.

PubMed

Brassard, M-E; Chouinard, P Y; Berthiaume, R; Tremblay, G F; Gervais, R; Martineau, R; Cinq-Mars, D

2015-11-01

Microbial protein synthesis in the rumen would be optimized when dietary carbohydrates and proteins have synchronized rates and extent of degradation. The aim of this study was to evaluate the effect of varying ruminal degradation rate of energy and nitrogen sources on intake, nitrogen balance, microbial protein yield, and kinetics of nutrients in the rumen of growing kids. Eight Boer goats (38.2 ± 3.0 kg) were used. The treatments were arranged in a split-plot Latin square design with grain sources (barley or corn) forming the main plots (squares). Grain processing methods and levels of protein degradability formed the subplots in a 2 × 2 factorial arrangement for a total of 8 dietary treatments. The grain processing method was rolling for barley and cracking for corn. Levels of protein degradability were obtained by feeding untreated soybean meal (SBM) or heat-treated soybean meal (HSBM). Each experimental period lasted 21 d, consisting of a 10-d adaptation period, a 7-d digestibility determination period, and a 4-d rumen evacuation and sampling period. Kids fed with corn had higher purine derivatives (PD) excretion when coupled with SBM compared with HSBM and the opposite occurred with barley-fed kids ( ≤ 0.01). Unprocessed grain offered with SBM led to higher PD excretion than with HSBM whereas protein degradability had no effect when processed grain was fed ( ≤ 0.03). Results of the current experiment with high-concentrate diets showed that microbial N synthesis could be maximized in goat kids by combining slowly fermented grains (corn or unprocessed grains) with a highly degradable protein supplement (SBM). With barley, a more rapidly fermented grain, a greater microbial N synthesis was observed when supplementing a low-degradable protein (HSBM).

Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana.

PubMed

Karuppanan, Kalimuthu; Duhra-Gill, Sifti; Kailemia, Muchena J; Phu, My L; Lebrilla, Carlito B; Dandekar, Abhaya M; Rodriguez, Raymond L; Nandi, Somen; McDonald, Karen A

2017-01-04

Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 ( CMG2 ) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus ( CaMV ) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N -glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N -glycans consists of complex type structures in both protein samples. The most abundant (>50%) N -glycan structure was GlcNAc₂(Xy l )Man₃(Fuc)GlcNAc₂ in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N -glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting.

Recognition deficits in mice carrying mutations of genes encoding BLOC-1 subunits pallidin or dysbindin.

PubMed

Spiegel, S; Chiu, A; James, A S; Jentsch, J D; Karlsgodt, K H

2015-11-01

Numerous studies have implicated DTNBP1, the gene encoding dystrobrevin-binding protein or dysbindin, as a candidate risk gene for schizophrenia, though this relationship remains somewhat controversial. Variation in dysbindin, and its location on chromosome 6p, has been associated with cognitive processes, including those relying on a complex system of glutamatergic and dopaminergic interactions. Dysbindin is one of the seven protein subunits that comprise the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Dysbindin protein levels are lower in mice with null mutations in pallidin, another gene in the BLOC-1, and pallidin levels are lower in mice with null mutations in the dysbindin gene, suggesting that multiple subunit proteins must be present to form a functional oligomeric complex. Furthermore, pallidin and dysbindin have similar distribution patterns in a mouse and human brain. Here, we investigated whether the apparent correspondence of pallid and dysbindin at the level of gene expression is also found at the level of behavior. Hypothesizing a mutation leading to underexpression of either of these proteins should show similar phenotypic effects, we studied recognition memory in both strains using the novel object recognition task (NORT) and social novelty recognition task (SNRT). We found that mice with a null mutation in either gene are impaired on SNRT and NORT when compared with wild-type controls. These results support the conclusion that deficits consistent with recognition memory impairment, a cognitive function that is impaired in schizophrenia, result from either pallidin or dysbindin mutations, possibly through degradation of BLOC-1 expression and/or function. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

A cytosolic copper storage protein provides a second level of copper tolerance in Streptomyces lividans.

PubMed

Straw, Megan L; Chaplin, Amanda K; Hough, Michael A; Paps, Jordi; Bavro, Vassiliy N; Wilson, Michael T; Vijgenboom, Erik; Worrall, Jonathan A R

2018-01-24

Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P 1 -type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Δccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance.

Analysis of Autoregulation at the Level of Pre-mRNA Splicing of the Suppressor-of-White-Apricot Gene in Drosophila

PubMed Central

Zachar, Z.; Chou, T. B.; Kramer, J.; Mims, I. P.; Bingham, P. M.

1994-01-01

The Drosophila suppressor-of-white-apricot [su(w(a))] protein regulates/modulates at least two somatic RNA processing events. It is a potent regulator of its own expression. We report here new studies of this autoregulatory circuit. Among other things, our studies show the following. First, new evidence that su(w(a)) expression is autoregulated at the level of pre-mRNA splicing is reported. su(w(a)) protein represses accumulation of the fully spliced su(w(a)) mRNA encoding it and promotes accumulation of high levels of incompletely spliced su(w(a)) pre-mRNA. Second, the fully spliced su(w(a)) mRNA is sufficient for all known su(w(a)) genetic functions indicating that it encodes the sole su(w(a)) protein. Third, the incompletely spliced su(w(a)) pre-mRNAs resulting from autoregulation are not translated (probably as a result of nuclear retention) and apparently represent nonfunctional by-products. Fourth, the special circumstances of su(w(a)) expression during oogenesis allows maternal deposition exclusively of fully spliced su(w(a)) mRNA. Fifth, su(w(a)) protein immunolocalizes to nuclei consistent with its being a direct regulator of pre-mRNA processing. We discuss the implications of our results for mechanisms of splicing regulation and for developmental control of su(w(a)) expression. PMID:8056305

Claudin gene expression patterns do not associate with interspecific differences in paracellular nutrient absorption.

PubMed

Price, Edwin R; Rott, Katherine H; Caviedes-Vidal, Enrique; Karasov, William H

2016-01-01

Bats exhibit higher paracellular absorption of glucose-sized molecules than non-flying mammals, a phenomenon that may be driven by higher permeability of the intestinal tight junctions. The various claudins, occludin, and other proteins making up the tight junctions are thought to determine their permeability properties. Here we show that absorption of the paracellular probe l-arabinose is higher in a bat (Eptesicus fuscus) than in a vole (Microtus pennsylvanicus) or a hedgehog (Atelerix albiventris). Furthermore, histological measurements demonstrated that hedgehogs have many more enterocytes in their intestines, suggesting that bats cannot have higher absorption of arabinose simply by having more tight junctions. We therefore investigated the mRNA levels of several claudins and occludin, because these proteins may affect permeability of tight junctions to macronutrients. To assess the expression levels of claudins per tight junction, we normalized the mRNA levels of the claudins to the constitutively expressed tight junction protein ZO-1, and combined these with measurements previously made in a bat and a rodent to determine if there were among-species differences. Although expression ratios of several genes varied among species, there was not a consistent difference between bats and non-flyers in the expression ratio of any particular gene. Protein expression patterns may differ from mRNA expression patterns, and might better explain differences among species in arabinose absorption. Copyright © 2015 Elsevier Inc. All rights reserved.

Mitochondrial Oxidative Phosphorylation Protein Levels in Peripheral Blood Mononuclear Cells Correlate with Levels in Subcutaneous Adipose Tissue within Samples Differing by HIV and Lipoatrophy Status

PubMed Central

Gerschenson, Mariana; Chow, Dominic; Libutti, Daniel E.; Willis, John H.; Murray, James; Capaldi, Roderick A.; Marusich, Michael

2008-01-01

Abstract Depletion of mitochondrial DNA (mtDNA) and mtDNA-encoded respiratory chain proteins in subcutaneous (SC) fat from patients with HIV lipoatrophy have clearly demonstrated the role of mitochondrial dysfunction in this syndrome. Research in HIV lipoatrophy, however, has been severely hampered by the lack of a suitable surrogate marker in blood or other easily obtained clinical specimens as fat biopsies are invasive and mtDNA levels in peripheral blood mononuclear cells (PBMC) do not consistently correlate with the disease process. We used a simple, rapid, quantitative 2-site dipstick immunoassay to measure OXPHOS enzymes Complex I (CI) and Complex IV (CIV), and rtPCR to measure mtDNA in 26 matched SC fat and PBMC specimens previously banked from individuals on potent antiretroviral (ARV) therapy with HIV lipoatrophy, on similar ARV therapy without lipoatrophy, and in HIV seronegative controls. Significant correlations were found between the respective PBMC and fat levels for both CI (r = 0.442, p = 0.024) and for CIV (r = 0.507, p = 0.008). Both CI and CIV protein levels were also significantly reduced in both PBMCs and fat in lipoatrophic subjects compared to HIV seronegative controls (p ≤ 0.05), while a comparative reduction in mtDNA levels in lipoatrophic subjects was observed only in fat. We conclude that CI and CIV levels in PBMCs correlate to their respective levels in fat and may have utility as surrogate markers of mitochondrial dysfunction in lipoatrophy. PMID:18844460

[Changes of protein tyrosine phosphorylation in erythrocyte band 3 glucose-6-phosphate dehydrogenase deficiency].

PubMed

Yu, Guoyu; Li, Jialin; Tian, Xingya; Lin, Hong; Wang, Xiaoying

2002-11-01

To explore the hemolytic mechanism of glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes in the view of phosphorylation of membrane protein. The alternation of membrane protein phosphorylation and the effect of dithiothreitol (DTT) on protein phosphorylation were analysed by Western blot technique. The activity of phosphotyrosine phosphatase (PTPs) was determined by using p-nitrophenyl phosphate as substrate. Tyrosine phosphorylation of band 3 protein was obviously enhanced in G6PD-deficient erythrocytes. The activity of PTPs was low compared to the normal erythrocytes. The level of phosphotyrosine in G6PD-deficient erythrocytes incubated with DTT was almost the same as in those without DTT. The results were consistent with the activity of PTPs. PTPs activity reduction and tyrosine phosphorylation enhancement induced by oxidation in G6PD deficiency play an important role in erythrocytes hemolysis. However, the alternation of thiol group is not the only factor affecting the activity of PTPs in G6PD-deficient erythrocytes.

The composition of cheetah (Acinonyx jubatus) milk.

PubMed

Osthoff, G; Hugo, A; de Wit, M

2006-01-01

Milk was obtained from two captive bred cheetahs. The nutrient content was 99.6 g protein; 64.8 g fat; and 40.21 g lactose per kg milk. Small amounts of oligosaccharides, glucose, galactose and fucose were noted. The protein fraction respectively consisted of 34.2 g caseins per kg milk and of 65.3 g whey proteins per kg milk. Very little variation in milk composition among the individual cheetahs was noted. Electrophoresis and identification of protein bands showed a similar migrating sequence of proteins as seen in lion's and cat's milk, with small differences in the beta-caseins. The lipid fraction contains 290.4 g saturated and 337.3 g mono-unsaturated fatty acids per kg milk fat respectively. The high content of 279.5 g kg(-1) milk fat of polyunsaturated fatty acids is due to a high content in alpha-linolenic acid. No short chain fatty acids, but substantial levels of uneven carbon chain fatty acids were observed.

The Neuronal Calcium Sensor Protein Acrocalcin: A Potential Target of Calmodulin Regulation during Development in the Coral Acropora millepora

PubMed Central

Reyes-Bermudez, Alejandro; Miller, David J.; Sprungala, Susanne

2012-01-01

To understand the calcium-mediated signalling pathways underlying settlement and metamorphosis in the Scleractinian coral Acropora millepora, a predicted protein set derived from larval cDNAs was scanned for the presence of EF-hand domains (Pfam Id: PF00036). This approach led to the identification of a canonical calmodulin (AmCaM) protein and an uncharacterised member of the Neuronal Calcium Sensor (NCS) family of proteins known here as Acrocalcin (AmAC). While AmCaM transcripts were present throughout development, AmAC transcripts were not detected prior to gastrulation, after which relatively constant mRNA levels were detected until metamorphosis and settlement. The AmAC protein contains an internal CaM-binding site and was shown to interact in vitro with AmCaM. These results are consistent with the idea that AmAC is a target of AmCaM in vivo, suggesting that this interaction may regulate calcium-dependent processes during the development of Acropora millepora. PMID:23284743

Serum Antibodies from a Subset of Horses Positive for Babesia caballi by Competitive Enzyme-Linked Immunosorbent Assay Demonstrate a Protein Recognition Pattern That Is Not Consistent with Infection

PubMed Central

Awinda, Peter O.; Mealey, Robert H.; Williams, Laura B. A.; Conrad, Patricia A.; Packham, Andrea E.; Reif, Kathryn E.; Grause, Juanita F.; Pelzel-McCluskey, Angela M.; Chung, Chungwon; Bastos, Reginaldo G.; Kappmeyer, Lowell S.; Howe, Daniel K.; Ness, SallyAnne L.; Knowles, Donald P.

2013-01-01

Tick-borne pathogens that cause persistent infection are of major concern to the livestock industry because of transmission risk from persistently infected animals and the potential economic losses they pose. The recent reemergence of Theileria equi in the United States prompted a widespread national survey resulting in identification of limited distribution of equine piroplasmosis (EP) in the U.S. horse population. This program identified Babesia caballi-seropositive horses using rhoptry-associated protein 1 (RAP-1)–competitive enzyme-linked immunosorbent assay (cELISA), despite B. caballi being considered nonendemic on the U.S. mainland. The purpose of the present study was to evaluate the suitability of RAP-1–cELISA as a single serological test to determine the infection status of B. caballi in U.S. horses. Immunoblotting indicated that sera from U.S. horses reacted with B. caballi lysate and purified B. caballi RAP-1 protein. Antibody reactivity to B. caballi lysate was exclusively directed against a single ∼50-kDa band corresponding to a native B. caballi RAP-1 protein. In contrast, sera from experimentally and naturally infected horses from regions where B. caballi is endemic bound multiple proteins ranging from 30 to 50 kDa. Dilutions of sera from U.S. horses positive by cELISA revealed low levels of antibodies, while sera from horses experimentally infected with B. caballi and from areas where B. caballi is endemic had comparatively high antibody levels. Finally, blood transfer from seropositive U.S. horses into naive horses demonstrated no evidence of B. caballi transmission, confirming that antibody reactivity in cELISA-positive U.S. horses was not consistent with infection. Therefore, we conclude that a combination of cELISA and immunoblotting is required for the accurate serodiagnosis of B. caballi. PMID:24049108

CAB-Align: A Flexible Protein Structure Alignment Method Based on the Residue-Residue Contact Area.

PubMed

Terashi, Genki; Takeda-Shitaka, Mayuko

2015-01-01

Proteins are flexible, and this flexibility has an essential functional role. Flexibility can be observed in loop regions, rearrangements between secondary structure elements, and conformational changes between entire domains. However, most protein structure alignment methods treat protein structures as rigid bodies. Thus, these methods fail to identify the equivalences of residue pairs in regions with flexibility. In this study, we considered that the evolutionary relationship between proteins corresponds directly to the residue-residue physical contacts rather than the three-dimensional (3D) coordinates of proteins. Thus, we developed a new protein structure alignment method, contact area-based alignment (CAB-align), which uses the residue-residue contact area to identify regions of similarity. The main purpose of CAB-align is to identify homologous relationships at the residue level between related protein structures. The CAB-align procedure comprises two main steps: First, a rigid-body alignment method based on local and global 3D structure superposition is employed to generate a sufficient number of initial alignments. Then, iterative dynamic programming is executed to find the optimal alignment. We evaluated the performance and advantages of CAB-align based on four main points: (1) agreement with the gold standard alignment, (2) alignment quality based on an evolutionary relationship without 3D coordinate superposition, (3) consistency of the multiple alignments, and (4) classification agreement with the gold standard classification. Comparisons of CAB-align with other state-of-the-art protein structure alignment methods (TM-align, FATCAT, and DaliLite) using our benchmark dataset showed that CAB-align performed robustly in obtaining high-quality alignments and generating consistent multiple alignments with high coverage and accuracy rates, and it performed extremely well when discriminating between homologous and nonhomologous pairs of proteins in both single and multi-domain comparisons. The CAB-align software is freely available to academic users as stand-alone software at http://www.pharm.kitasato-u.ac.jp/bmd/bmd/Publications.html.

Zn-metalloprotease sequences in extremophiles

NASA Astrophysics Data System (ADS)

Holden, T.; Dehipawala, S.; Golebiewska, U.; Cheung, E.; Tremberger, G., Jr.; Williams, E.; Schneider, P.; Gadura, N.; Lieberman, D.; Cheung, T.

2010-09-01

The Zn-metalloprotease family contains conserved amino acid structures such that the nucleotide fluctuation at the DNA level would exhibit correlated randomness as described by fractal dimension. A nucleotide sequence fractal dimension can be calculated from a numerical series consisting of the atomic numbers of each nucleotide. The structure's vibration modes can also be studied using a Gaussian Network Model. The vibration measure and fractal dimension values form a two-dimensional plot with a standard vector metric that can be used for comparison of structures. The preference for amino acid usage in extremophiles may suppress nucleotide fluctuations that could be analyzed in terms of fractal dimension and Shannon entropy. A protein level cold adaptation study of the thermolysin Zn-metalloprotease family using molecular dynamics simulation was reported recently and our results show that the associated nucleotide fluctuation suppression is consistent with a regression pattern generated from the sequences's fractal dimension and entropy values (R-square { 0.98, N =5). It was observed that cold adaptation selected for high entropy and low fractal dimension values. Extension to the Archaemetzincin M54 family in extremophiles reveals a similar regression pattern (R-square = 0.98, N = 6). It was observed that the metalloprotease sequences of extremely halophilic organisms possess high fractal dimension and low entropy values as compared with non-halophiles. The zinc atom is usually bonded to the histidine residue, which shows limited levels of vibration in the Gaussian Network Model. The variability of the fractal dimension and entropy for a given protein structure suggests that extremophiles would have evolved after mesophiles, consistent with the bias usage of non-prebiotic amino acids by extremophiles. It may be argued that extremophiles have the capacity to offer extinction protection during drastic changes in astrobiological environments.

Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues.

PubMed

Banks, Rosamonde E; Craven, Rachel A; Harnden, Patricia A; Selby, Peter J

2003-04-01

Western blotting remains a central technique in confirming identities of proteins, their quantitation and analysis of various isoforms. The biotin-avidin/streptavidin system is often used as an amplification step to increase sensitivity but in some tissues such as kidney, "nonspecific" interactions may be a problem due to high levels of endogenous biotin-containing proteins. The EnVision system, developed for immunohistochemical applications, relies on binding of a polymeric conjugate consisting of up to 100 peroxidase molecules and 20 secondary antibody molecules linked directly to an activated dextran backbone, to the primary antibody. This study demonstrates that it is also a viable and sensitive alternative detection system in Western blotting applications.

Large-scale FMO-MP3 calculations on the surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA)

NASA Astrophysics Data System (ADS)

Mochizuki, Yuji; Yamashita, Katsumi; Fukuzawa, Kaori; Takematsu, Kazutomo; Watanabe, Hirofumi; Taguchi, Naoki; Okiyama, Yoshio; Tsuboi, Misako; Nakano, Tatsuya; Tanaka, Shigenori

2010-06-01

Two proteins on the influenza virus surface have been well known. One is hemagglutinin (HA) associated with the infection to cells. The fragment molecular orbital (FMO) calculations were performed on a complex consisting of HA trimer and two Fab-fragments at the third-order Møller-Plesset perturbation (MP3) level. The numbers of residues and 6-31G basis functions were 2351 and 201276, and thus a massively parallel-vector computer was utilized to accelerate the processing. This FMO-MP3 job was completed in 5.8 h with 1024 processors. Another protein is neuraminidase (NA) involved in the escape from infected cells. The FMO-MP3 calculation was also applied to analyze the interactions between oseltamivir and surrounding residues in pharmacophore.

The transcriptomic fingerprint of glucoamylase over-expression in Aspergillus niger

PubMed Central

2012-01-01

Background Filamentous fungi such as Aspergillus niger are well known for their exceptionally high capacity for secretion of proteins, organic acids, and secondary metabolites and they are therefore used in biotechnology as versatile microbial production platforms. However, system-wide insights into their metabolic and secretory capacities are sparse and rational strain improvement approaches are therefore limited. In order to gain a genome-wide view on the transcriptional regulation of the protein secretory pathway of A. niger, we investigated the transcriptome of A. niger when it was forced to overexpression the glaA gene (encoding glucoamylase, GlaA) and secrete GlaA to high level. Results An A. niger wild-type strain and a GlaA over-expressing strain, containing multiple copies of the glaA gene, were cultivated under maltose-limited chemostat conditions (specific growth rate 0.1 h-1). Elevated glaA mRNA and extracellular GlaA levels in the over-expressing strain were accompanied by elevated transcript levels from 772 genes and lowered transcript levels from 815 genes when compared to the wild-type strain. Using GO term enrichment analysis, four higher-order categories were identified in the up-regulated gene set: i) endoplasmic reticulum (ER) membrane translocation, ii) protein glycosylation, iii) vesicle transport, and iv) ion homeostasis. Among these, about 130 genes had predicted functions for the passage of proteins through the ER and those genes included target genes of the HacA transcription factor that mediates the unfolded protein response (UPR), e.g. bipA, clxA, prpA, tigA and pdiA. In order to identify those genes that are important for high-level secretion of proteins by A. niger, we compared the transcriptome of the GlaA overexpression strain of A. niger with six other relevant transcriptomes of A. niger. Overall, 40 genes were found to have either elevated (from 36 genes) or lowered (from 4 genes) transcript levels under all conditions that were examined, thus defining the core set of genes important for ensuring high protein traffic through the secretory pathway. Conclusion We have defined the A. niger genes that respond to elevated secretion of GlaA and, furthermore, we have defined a core set of genes that appear to be involved more generally in the intensified traffic of proteins through the secretory pathway of A. niger. The consistent up-regulation of a gene encoding the acetyl-coenzyme A transporter suggests a possible role for transient acetylation to ensure correct folding of secreted proteins. PMID:23237452

Level of supplemental protein does not influence the ruminally undegradable protein value.

PubMed

Legleiter, L R; Mueller, A M; Kerley, M S

2005-04-01

Two experiments were conducted to determine whether elevating the percentage of ruminally undegradable protein (RUP) in the diet would influence the RUP value of the protein feedstuff. A single-effluent, continuous-culture study was designed to test the effect of RUP inclusion rate in the diet on ruminal degradability of the protein. Treatments consisted (DM basis) of a control diet with no supplemental protein, control + 2.5% bloodmeal (BM-L), control + 5% bloodmeal (BM-H), control + 4.45% soybean meal (SBM-L), and control + 8.89% soybean meal (SBM-H). Proteolytic activity and total VFA concentration were not affected (P = 0.73 and P = 0.13) by treatment. Within protein source, dietary RUP value was not affected (P = 0.94) by level of inclusion. When corrected for control diet RUP flow, the RUP value of the blood meal (BM) protein was higher (P = 0.01) than soybean meal (SBM); however, level of supplementation did not affect (P = 0.07) the RUP value of BM or SBM. In Exp. 2, 32 British x Continental crossbred steers (276 +/- 26.3 kg) were fed for 72 d to examine the effects of balancing the AA:energy ratio, using BM as a RUP source, on ADG, G:F, and lean tissue deposition. Diets were formulated to provide increasing levels of arginine, while ruminally degradable protein and energy were held constant. Four dietary treatments provided 0.5, 1, 1.5, and 2x the required amount of arginine, whereas the control diet had no BM included. Daily DMI averaged 7.6 kg/steer and did not differ (P = 0.71) among treatments. Steers gained an average of 1.9 kg/d and average G:F was 0.260, with no differences (P = 0.60 and P = 0.97, respectively) among treatments. There was no difference (P = 0.48) in the change in 12th-rib fat depth during the study; however, change in LM area was affected quadratically as the level of BM increased in the diet, with the greatest increase in LM area occurring in steers fed the 1x and 1.5x required arginine treatments. Balancing the AA:energy ratio did not affect G:F, DMI, or ADG; however, it increased deposition of lean in the LM quadratically. Level of dietary inclusion of BM as an RUP source does not affect its RUP value or efficacy of providing postruminal AA in growing steers.

Modification and optimization of the united-residue (UNRES) potential-energy function for canonical simulations. I. Temperature dependence of the effective energy function and tests of the optimization method with single training proteins

PubMed Central

Liwo, Adam; Khalili, Mey; Czaplewski, Cezary; Kalinowski, Sebastian; Ołdziej, Stanisław; Wachucik, Katarzyna; Scheraga, Harold A.

2011-01-01

We report the modification and parameterization of the united-residue (UNRES) force field for energy-based protein-structure prediction and protein-folding simulations. We tested the approach on three training proteins separately: 1E0L (β), 1GAB (α), and 1E0G (α + β). Heretofore, the UNRES force field had been designed and parameterized to locate native-like structures of proteins as global minima of their effective potential-energy surfaces, which largely neglected the conformational entropy because decoys composed of only lowest-energy conformations were used to optimize the force field. Recently, we developed a mesoscopic dynamics procedure for UNRES, and applied it with success to simulate protein folding pathways. How ever, the force field turned out to be largely biased towards α-helical structures in canonical simulations because the conformational entropy had been neglected in the parameterization. We applied the hierarchical optimization method developed in our earlier work to optimize the force field, in which the conformational space of a training protein is divided into levels each corresponding to a certain degree of native-likeness. The levels are ordered according to increasing native-likeness; level 0 corresponds to structures with no native-like elements and the highest level corresponds to the fully native-like structures. The aim of optimization is to achieve the order of the free energies of levels, decreasing as their native-likeness increases. The procedure is iterative, and decoys of the training protein(s) generated with the energy-function parameters of the preceding iteration are used to optimize the force field in a current iteration. We applied the multiplexing replica exchange molecular dynamics (MREMD) method, recently implemented in UNRES, to generate decoys; with this modification, conformational entropy is taken into account. Moreover, we optimized the free-energy gaps between levels at temperatures corresponding to a predominance of folded or unfolded structures, as well as to structures at the putative folding-transition temperature, changing the sign of the gaps at the transition temperature. This enabled us to obtain force fields characterized by a single peak in the heat capacity at the transition temperature. Furthermore, we introduced temperature dependence to the UNRES force field; this is consistent with the fact that it is a free-energy and not a potential-energy function. PMID:17201450

A nine-country study of the protein content and amino acid composition of mature human milk

PubMed Central

Feng, Ping; Gao, Ming; Burgher, Anita; Zhou, Tian Hui; Pramuk, Kathryn

2016-01-01

Background Numerous studies have evaluated protein and amino acid levels in human milk. However, research in this area has been limited by small sample sizes and study populations with little ethnic or racial diversity. Objective Evaluate the protein and amino acid composition of mature (≥30 days) human milk samples collected from a large, multinational study using highly standardized methods for sample collection, storage, and analysis. Design Using a single, centralized laboratory, human milk samples from 220 women (30–188 days postpartum) from nine countries were analyzed for amino acid composition using Waters AccQ-Tag high-performance liquid chromatography and total nitrogen content using the LECO FP-528 nitrogen analyzer. Total protein was calculated as total nitrogen×6.25. True protein, which includes protein, free amino acids, and peptides, was calculated from the total amino acids. Results Mean total protein from individual countries (standard deviation [SD]) ranged from 1,133 (125.5) to 1,366 (341.4) mg/dL; the mean across all countries (SD) was 1,192 (200.9) mg/dL. Total protein, true protein, and amino acid composition were not significantly different across countries except Chile, which had higher total and true protein. Amino acid profiles (percent of total amino acids) did not differ across countries. Total and true protein concentrations and 16 of 18 amino acid concentrations declined with the stage of lactation. Conclusions Total protein, true protein, and individual amino acid concentrations in human milk steadily decline from 30 to 151 days of lactation, and are significantly higher in the second month of lactation compared with the following 4 months. There is a high level of consistency in the protein content and amino acid composition of human milk across geographic locations. The size and diversity of the study population and highly standardized procedures for the collection, storage, and analysis of human milk support the validity and broad application of these findings. PMID:27569428

Time course for the modulation of hepatic cytochrome P450 after administration of ethylbenzene and its correlation with toluene metabolism.

PubMed

Yuan, W; Sequeira, D J; Cawley, G F; Eyer, C S; Backes, W L

1997-03-01

The goal of the present study was to examine the time course for changes in P450 expression and hydrocarbon metabolism after acute treatment with the simple aromatic hydrocarbon ethylbenzene (EB) and to correlate these alterations with the changes observed in alkylbenzene metabolism. Male Holtzman rats were treated with a single intraperitoneal injection of EB, and the effects on specific P450-dependent activities, immunoreactive P450 isozyme levels, and RNA levels were measured at various times after injection. Toluene was used as the test alkylbenzene for examination of the EB-mediated changes on in vitro hydrocarbon metabolism. In untreated rats, toluene was metabolized almost entirely by aliphatic hydroxylation (to benzyl alcohol); however, in EB-treated rats, significant quantities of benzyl alcohol, o-cresol, and p-cresol were produced. Interestingly, 5-10 h after EB treatment, there was a 40% decrease in benzyl alcohol production. By 24 h, rates of benzyl alcohol formation returned to control levels, whereas there was a 7-fold increase in o-cresol and a greater that 50-fold increase in p-cresol production. The changes in the disposition of toluene were then correlated with changes in particular P450 isozymes. Several P450 isozymes were induced after EB administration. P450 2B1/2-dependent testosterone 16 beta-hydroxylation and P450 2B1/2-immunoreactive protein were elevated 30-fold after EB administration, reaching maxima by 24 h and remaining elevated 48 h after exposure. Changes in P450 2B1 and 2B2 RNA preceded those of the proteins. Similar results were observed with P450 1A1. P450 2E1 RNA levels were elevated after a single EB injection. However, the elevation in P450 2E1-dependent activities and immunoreactive protein levels preceded the changes in RNA, suggesting that multiple steps are affected by EB exposure. In contrast to the increases in some isozymes, P450 2C11 protein was rapidly suppressed (within the first 2-10 h) after hydrocarbon exposure, suggestive of a destabilization of the protein. When comparing the changes in P450 isozymes to alterations in toluene metabolism, the immediate suppression in aliphatic hydroxylation of toluene (in the first 5-10 h) was consistent with the decrease in P450 2C11. Subsequent to this effect, P450 2B1/2 and 2E1 were induced, which elevated production of this metabolite to control levels. The increase in the aromatic hydroxylation of toluene to both o, and p-cresol was consistent with the induction of P450s 2B1/2, 2E1, and 1A1.

Biologically active LIL proteins built with minimal chemical diversity

PubMed Central

Heim, Erin N.; Marston, Jez L.; Federman, Ross S.; Edwards, Anne P. B.; Karabadzhak, Alexander G.; Petti, Lisa M.; Engelman, Donald M.; DiMaio, Daniel

2015-01-01

We have constructed 26-amino acid transmembrane proteins that specifically transform cells but consist of only two different amino acids. Most proteins are long polymers of amino acids with 20 or more chemically distinct side-chains. The artificial transmembrane proteins reported here are the simplest known proteins with specific biological activity, consisting solely of an initiating methionine followed by specific sequences of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a methyl group. We designate these proteins containing leucine (L) and isoleucine (I) as LIL proteins. These proteins functionally interact with the transmembrane domain of the platelet-derived growth factor β-receptor and specifically activate the receptor to transform cells. Complete mutagenesis of these proteins identified individual amino acids required for activity, and a protein consisting solely of leucines, except for a single isoleucine at a particular position, transformed cells. These surprisingly simple proteins define the minimal chemical diversity sufficient to construct proteins with specific biological activity and change our view of what can constitute an active protein in a cellular context. PMID:26261320

Possible role of interference, protein noise, and sink effects in nonphotochemical quenching in photosynthetic complexes.

PubMed

Berman, Gennady P; Nesterov, Alexander I; Gurvitz, Shmuel; Sayre, Richard T

2017-01-01

We analyze theoretically a simple and consistent quantum mechanical model that reveals the possible role of quantum interference, protein noise, and sink effects in the nonphotochemical quenching (NPQ) in light-harvesting complexes (LHCs). The model consists of a network of five interconnected sites (excitonic states of light-sensitive molecules) responsible for the NPQ mechanism. The model also includes the "damaging" and the dissipative channels. The damaging channel is responsible for production of singlet oxygen and other destructive outcomes. In our model, both damaging and "dissipative" charge transfer channels are described by discrete electron energy levels attached to their sinks, that mimic the continuum part of electron energy spectrum. All five excitonic sites interact with the protein environment that is modeled using a stochastic process. Our approach allowed us to derive the exact and closed system of linear ordinary differential equations for the reduced density matrix and its first momentums. These equations are solved numerically including for strong interactions between the light-sensitive molecules and protein environment. As an example, we apply our model to demonstrate possible contributions of quantum interference, protein noise, and sink effects in the NPQ mechanism in the CP29 minor LHC. The numerical simulations show that using proper combination of quantum interference effects, properties of noise, and sinks, one can significantly suppress the damaging channel. Our findings demonstrate the possible role of interference, protein noise, and sink effects for modeling, engineering, and optimizing the performance of the NPQ processes in both natural and artificial light-harvesting complexes.

Possible role of interference, protein noise, and sink effects in nonphotochemical quenching in photosynthetic complexes

DOE PAGES

Berman, Gennady P.; Nesterov, Alexander I.; Gurvitz, Shmuel; ...

2016-04-30

Here, we analyze theoretically a simple and consistent quantum mechanical model that reveals the possible role of quantum interference, protein noise, and sink effects in the nonphotochemical quenching (NPQ) in light-harvesting complexes (LHCs). The model consists of a network of five interconnected sites (excitonic states of light-sensitive molecules) responsible for the NPQ mechanism. The model also includes the “damaging” and the dissipative channels. The damaging channel is responsible for production of singlet oxygen and other destructive outcomes. In this model, both damaging and “dissipative” charge transfer channels are described by discrete electron energy levels attached to their sinks, that mimicmore » the continuum part of electron energy spectrum. All five excitonic sites interact with the protein environment that is modeled using a stochastic process. Our approach allowed us to derive the exact and closed system of linear ordinary differential equations for the reduced density matrix and its first momentums. Moreover, these equations are solved numerically including for strong interactions between the light-sensitive molecules and protein environment. As an example, we apply our model to demonstrate possible contributions of quantum interference, protein noise, and sink effects in the NPQ mechanism in the CP29 minor LHC. The numerical simulations show that using proper combination of quantum interference effects, properties of noise, and sinks, one can significantly suppress the damaging channel. Finally, our findings demonstrate the possible role of interference, protein noise, and sink effects for modeling, engineering, and optimizing the performance of the NPQ processes in both natural and artificial light-harvesting complexes.« less

Differential expression proteins associated with bud dormancy release during chilling treatment of tree peony (Paeonia suffruticosa).

PubMed

Zhang, Y X; Yu, D; Tian, X L; Liu, C Y; Gai, S P; Zheng, G S

2015-01-01

Endo-dormant flower buds of tree peony must have sufficient chilling duration to reinitiate growth, which is a major obstacle to the forcing culture of tree peony in winter. We used a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS) to identify the differentially expressed proteins of tree peony after three different chilling treatments: endo-dormancy, endo-dormancy release and eco-dormancy stages. More than 200 highly reproducible protein spots were detected, and 31 differentially expressed spots (P < 0.05) were selected for further analysis. Finally, 20 protein spots were confidently identified from databases, which were annotated and classified into seven functional categories: response to abiotic or biotic stimulus (four), metabolic processes (four), other binding (three), transcription or transcription regulation (two), biological processes (one), cell biogenesis (one) and unclassified (five). The results of qPCR of five genes were mainly consistent with that of the protein accumulation analysis as determined by 2-DE. This indicated that most of these genes were mainly regulated at transcriptional level. The activity of nitrate reductase and pyruvate dehydrogenase E1 was consistent with the 2-DE results. The proteomic profiles indicated activation of citrate cycle, amino acid metabolism, lipid metabolism, energy production, calcium signalling and cell growth processes by chilling fulfilment to facilitate dormancy release in tree peony. Analysis of functions of identified proteins will increase our knowledge of endo-dormancy release in tree peony. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Mechanical stability analysis of the protein L immunoglobulin-binding domain by full alanine screening using molecular dynamics simulations.

PubMed

Glyakina, Anna V; Likhachev, Ilya V; Balabaev, Nikolay K; Galzitskaya, Oxana V

2015-03-01

This article is the first to study the mechanical properties of the immunoglobulin-binding domain of protein L (referred to as protein L) and its mutants at the atomic level. In the structure of protein L, each amino acid residue (except for alanines and glycines) was replaced sequentially by alanine. Thus, 49 mutants of protein L were obtained. The proteins were stretched at their termini at constant velocity using molecular dynamics simulations in water, i.e. by forced unfolding. 19 out of 49 mutations resulted in a large decrease of mechanical protein stability. These amino acids were affecting either the secondary structure (11 mutations) or loop structures (8 mutations) of protein L. Analysis of mechanical unfolding of the generated protein that has the same topology as protein L but consists of only alanines and glycines allows us to suggest that the mechanical stability of proteins, and specifically protein L, is determined by interactions between certain amino acid residues, although the unfolding pathway depends on the protein topology. This insight can now be used to modulate the mechanical properties of proteins and their unfolding pathways in the desired direction for using them in various biochips, biosensors and biomaterials for medicine, industry, and household purposes. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Study on proteomics of familial systemic lupus erythematosus patients in one family from Sichuan, China].

PubMed

Wu, Yong-kang; Huang, Zhuo-chun; Shi, Yun-ying; Cai, Bei; Wang, Lan-lan; Ying, Bin-wu; Hu, Chao-jun; Li, Yong-zhe

2009-05-01

To investigate the proteomic characteristics of systemic lupus erythematosus (SLE) in a SLE family from Sichuan, China which consisting of 7 members with 3 SLE cases, and to find the proteins correlated with the heredity of SLE. A total of 153 serum samples were collected from 7 members including 3 SLE sisters in this SLE family, 63 individual SLE patients, as well as 83 healthy controls. The diagnosis of SLE is based on the American College of Rheumatology criteria (1997). All serum samples were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with magnetic beads technology. Serum protein profiles were obtained by MALDI-TOF-MS combined with magnetic beads in order to identify predictive biomarkers of risk of suffering SLE. The resulting spectra were analyzed with Biomarker Wizard software 3.1.0. Four discriminative mass/charge (m/z) proteins serving as pathogenic biomarkers were identified on arrays for family SLE cases versus individual SLE and healthy controls. The protein level of peak intensities at m/z of 9342.23 was significantly greater in SLE family group compared with that in individual SLE patients and healthy controls (P<0.05), those of individual SLE patients were significantly greater compared with healthy controls (P<0.05); the proteins level of peak intensities at m/z of 4094.03, 5905.35 and 7973.53 in SLE family group were significantly lower compared with that in individual SLE patients and healthy controls (P<0.05), those of individual SLE patients were significantly lower compared with healthy controls (P<0.05). The proteins of m/z of 9342.23, 4094.03, 5905.35 and 7973.53 maybe play a great role in assemble pathogenesis of SLE and predict the risk of suffering SLE. The higher protein level of m/z of 9342.23 and the lower protein level of m/z of 4094.03, 5905.35 and 7973.53, the higher risk of sufferring with SLE.

Direct observation of transcription activator-like effector (TALE) protein dynamics

NASA Astrophysics Data System (ADS)

Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M.

2014-03-01

In this work, we describe a single molecule assay to probe the site-search dynamics of transcription activator-like effector (TALE) proteins along DNA. In modern genetics, the ability to selectively edit the human genome is an unprecedented development, driven by recent advances in targeted nuclease proteins. Specific gene editing can be accomplished using TALE proteins, which are programmable DNA-binding proteins that can be fused to a nuclease domain. In this way, TALENs are a leading technology that has shown great success in the genomic editing of pluripotent stem cells. A major hurdle facing clinical implementation, however, is the potential for deleterious off-target binding events. For these reasons, a molecular-level understanding of TALE binding and target sequence search on DNA is essential. To this end, we developed a single-molecule fluorescence imaging assay that provides a first-of-its-kind view of the 1-D diffusion of TALE proteins along stretched DNA. Taken together with co-crystal structures of DNA-bound TALEs, our results suggest a rotationally-coupled, major groove tracking model for diffusion. We further report diffusion constants for TALE proteins as a function of salt concentration, consistent with previously described models of 1-D protein diffusion.

Structural perturbation of proteins in low denaturant concentrations.

PubMed

Basak, S; Debnath, D; Haque, E; Ray, S; Chakrabarti, A

2001-01-01

The presence of very low concentrations of the widely used chemical denaturants, guanidinium chloride and urea, induce changes in the tertiary structure of proteins. We have presented results on such changes in four structurally unrelated proteins to show that such structural perturbations are common irrespective of their origin. Data representative of such structural changes are shown for the monomeric globular proteins such as horseradish peroxidase (HRP) from a plant, human serum albumin (HSA) and prothrombin from ovine blood serum, and for the membrane-associated, worm-like elongated protein, spectrin, from ovine erythrocytes. Structural alterations in these proteins were reflected in quenching studies of tryptophan fluorescence using the widely used quencher acrylamide. Stern-Volmer quenching constants measured in presence of the denaturants, even at concentrations below 100 mM, were higher than those measured in absence of the denaturants. Both steady-state and time-resolved fluorescence emission properties of tryptophan and of the extrinsic probe PRODAN were used for monitoring conformational changes in the proteins in presence of different low concentrations of the denaturants. These results are consistent with earlier studies from our laboratory indicating structural perturbations in proteins at the tertiary level, keeping their native-like secondary structure and their biological activity more or less intact.

Running-Induced Systemic Cathepsin B Secretion Is Associated with Memory Function.

PubMed

Moon, Hyo Youl; Becke, Andreas; Berron, David; Becker, Benjamin; Sah, Nirnath; Benoni, Galit; Janke, Emma; Lubejko, Susan T; Greig, Nigel H; Mattison, Julie A; Duzel, Emrah; van Praag, Henriette

2016-08-09

Peripheral processes that mediate beneficial effects of exercise on the brain remain sparsely explored. Here, we show that a muscle secretory factor, cathepsin B (CTSB) protein, is important for the cognitive and neurogenic benefits of running. Proteomic analysis revealed elevated levels of CTSB in conditioned medium derived from skeletal muscle cell cultures treated with AMP-kinase agonist AICAR. Consistently, running increased CTSB levels in mouse gastrocnemius muscle and plasma. Furthermore, recombinant CTSB application enhanced expression of brain-derived neurotrophic factor (BDNF) and doublecortin (DCX) in adult hippocampal progenitor cells through a mechanism dependent on the multifunctional protein P11. In vivo, in CTSB knockout (KO) mice, running did not enhance adult hippocampal neurogenesis and spatial memory function. Interestingly, in Rhesus monkeys and humans, treadmill exercise elevated CTSB in plasma. In humans, changes in CTSB levels correlated with fitness and hippocampus-dependent memory function. Our findings suggest CTSB as a mediator of effects of exercise on cognition. Published by Elsevier Inc.

Requirement for tryptophan by milkfish (Chanos chanos Forsskal) juveniles.

PubMed

Coloso, R M; Tiro, L B; Benitez, L V

1992-05-01

Groups of milkfish juveniles (mean initial weight 7.7 g) were fed semipurified diets containing 0.9, 1.4, 2.1, 3.1, 4.1 and 6.1 g tryptophan/kg dry diet for 12 weeks. The mean crude protein content of the diets (containing white fishmeal, gelatin and free amino acid mixture to simulate the pattern of hydrolysed milkfish protein) was 49%. On the basis of the growth response, the tryptophan requirement of milkfish juveniles was estimated to be 3.1 g/kg diet. Fish fed low levels of tryptophan exhibited low weight gains and poor feed conversion ratios. Survival (92-100%) was consistently high in all treatments. Fish fed diets containing tryptophan levels greater than 3.1 g/kg had slightly lower survival rates. The activity of hepatic tryptophan pyrrolase showed no significant differences with increasing dietary tryptophan levels. No nutritional deficiency signs were observed other than the depression in growth rates in fish given the tryptophan deficient diets.

Altered expressions of apoptotic factors and synaptic markers in postmortem brain from bipolar disorder patients

PubMed Central

Kim, Hyung-Wook; Rapoport, Stanley I; Rao, Jagadeesh S

2009-01-01

Bipolar disorder (BD) is a progressive psychiatric disorder characterized by recurrent changes of mood, and is associated with cognitive decline. There is evidence of excitotoxicity, neuroinflammation, upregulated arachidonic acid (AA) cascade signaling and brain atrophy in BD patients. These observations suggest that BD pathology may be associated with apoptosis as well as with disturbed synaptic function. To test this hypothesis, we measured mRNA and protein levels of the pro-apoptotic (Bax, BAD, Caspase-9 and Caspase-3) and anti-apoptotic factors (BDNF and Bcl-2), and of pre- and post-synaptic markers (synaptophysin and drebrin), in postmortem brain from 10 BD patients and 10 age-matched controls. Consistent with the hypothesis, BD brains showed significant increases in protein and mRNA levels of the pro-apoptotic factors and significant decreases of levels of the anti-apoptotic factors and the synaptic markers, synaptophysin and drebrin. These differences may contribute to brain atrophy and progressive cognitive changes in BD. PMID:19945534

Modelling atherosclerosis by proteomics: Molecular changes in the ascending aortas of cholesterol-fed rabbits.

PubMed

Xu, Jingshu; Jüllig, Mia; Middleditch, Martin J; Cooper, Garth J S

2015-09-01

The cholesterol-fed rabbit is commonly used as a model to study the vascular effects of hypercholesterolemia and resulting atherosclerotic lesions. Here we undertook a proteomic case-control investigation of ascending aortas from male New Zealand White rabbits after 10 weeks on a high-cholesterol (2% w/w) diet (HCD, n = 5) or control diet (n = 5), in order to determine the changes in response to the HCD. Histology confirmed intimal thickening in the HCD group consistent with atherosclerosis, and LC-MS/MS analysis of individually-obtained ascending aortic extracts labelled with isobaric (iTRAQ) tags enabled the identification and quantitation of 453 unique proteins above the 1% false discovery rate threshold. Of 67 proteins showing significant differences in relative abundance (p < 0.05), 62 were elevated and five decreased in ascending aortas from HCD-fed rabbits compared to controls. Six proteins were selected for validation using Multiple Reaction Monitoring, which confirmed the iTRAQ results. Many of the observed protein changes are consistent with known molecular perturbations in the ascending aorta that occur in response to hypercholesterolemia, e.g. elevation of tissue levels of apolipoproteins, extracellular matrix adhesion proteins, glycolytic enzymes, heat shock proteins and proteins involved in immune defense. We also made a number of novel observations, including a 15-fold elevation of glycoprotein (trans-membrane) nmb-like (Gpnmb) in response to HCD. Gpnmb has previously been linked to angiogenesis but not to atherosclerosis. This and additional novel observations merit further investigation as these perturbations may play important and as yet undiscovered roles in the pathogenesis of atherosclerosis in rabbits as well as humans. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity

PubMed Central

Ma, Pikyee; Patching, Simon G.; Ivanova, Ekaterina; Baldwin, Jocelyn M.; Sharples, David; Baldwin, Stephen A.

2016-01-01

This work reports the evolutionary relationships, amplified expression, functional characterization and purification of the putative allantoin transport protein, PucI, from Bacillus subtilis. Sequence alignments and phylogenetic analysis confirmed close evolutionary relationships between PucI and membrane proteins of the nucleobase-cation-symport-1 family of secondary active transporters. These include the sodium-coupled hydantoin transport protein, Mhp1, from Microbacterium liquefaciens, and related proteins from bacteria, fungi and plants. Membrane topology predictions for PucI were consistent with 12 putative transmembrane-spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. The pucI gene was cloned into the IPTG-inducible plasmid pTTQ18 upstream from an in-frame hexahistidine tag and conditions determined for optimal amplified expression of the PucI(His6) protein in Escherichia coli to a level of about 5 % in inner membranes. Initial rates of inducible PucI-mediated uptake of 14C-allantoin into energized E. coli whole cells conformed to Michaelis–Menten kinetics with an apparent affinity (K mapp) of 24 ± 3 μM, therefore confirming that PucI is a medium-affinity transporter of allantoin. Dependence of allantoin transport on sodium was not apparent. Competitive uptake experiments showed that PucI recognizes some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides. PucI(His6) was solubilized from inner membranes using n-dodecyl-β-d-maltoside and purified. The isolated protein contained a substantial proportion of α-helix secondary structure, consistent with the predictions, and a 3D model was therefore constructed on a template of the Mhp1 structure, which aided localization of the potential ligand binding site in PucI. PMID:26967546

A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts.

PubMed

Richter, Lubna V; Yang, Huijun; Yazdani, Mohammad; Hanson, Maureen R; Ahner, Beth A

2018-01-01

We investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production. The downstream box (DB) of the TetC or NPTII genes, the first 15 codons following the start codon, was N -terminally fused to the coding region of cel6A , an endoglucanase from Thermobifida fusca . We also employed a chimeric regulatory element, consisting of the 16S rRNA promoter and the atpA 5'UTR, previously reported to enhance protein expression, to regulate the expression of the TetC- cel6A gene. We further investigated the accumulation of TetC-Cel6A under N -deplete growth conditions. Both of the DB fusions improved intracellular accumulation of Cel6A in transplastomic C. reinhardtii strains though the TetC DB was much more effective than the NPTII DB. Furthermore, using the chimeric regulatory element, the TetC-Cel6A protein accumulation displayed a significant increase to 0.3% total soluble protein (TSP), whereas NPTII-Cel6A remained too low to quantify. Comparable levels of TetC- and NPTII- cel6A transcripts were observed, which suggests that factors other than transcript abundance mediate the greater TetC-Cel6A accumulation. The TetC-Cel6A accumulation was stable regardless of the growth stage, and the transplastomic strain growth rate was not altered. When transplastomic cells were suspended in N -deplete medium, cellular levels of TetC-Cel6A increased over time along with TSP, and were greater than those in cells suspended in N -replete medium. The DB fusion holds great value as a tool to enhance foreign protein accumulation in C. reinhardtii chloroplasts and its influence is related to translation or other post-transcriptional processes. Our results also suggest that transplastomic protein production can be compatible with algal biofuel production strategies. Cells displayed a consistent accumulation of recombinant protein throughout the growth phase and nitrogen starvation, a strategy used to induce lipid production in algae, led to higher cellular heterologous protein content. The latter result is contrary to what might have been expected a priori and is an important result for the development of future algal biofuel systems, which will likely require co-products for economic sustainability.

Materials for Fluorescence Resonance Energy Transfer Analysis: Beyond Traditional Donor-Acceptor Combinations

DTIC Science & Technology

2006-01-01

a variety of surface functionalities (e.g., biotin, avidin, collagen , amines, alde- hydes, sulfates, and carboxylates) that allow facile bioconju...relative expression level.[14,20,21] The green fluorescent protein (GFP; Figure 9) derived from the jellyfish Aequorea victoria is the prototypical...derived from jellyfish , is a Ca2+-sensitive bioluminescent photoprotein consisting of the luciferase apoaequorin complexed to its coelenterazine

Influence of reproductive tract obstruction on expression of epididymal proteins and their restoration after patency.

PubMed

Li, Bing-Kun; Wang, Xiang; Liu, Chun-Xiao; Zheng, Shao-Bo; Li, Hu-Lin; Li, Li-Ping; Xu, A-Bai

2013-01-01

Vasectomy is a simple and reliable method of male contraception. A growing number of men after vasectomy request vasectomy reversal due to various reasons. The pregnancy rate is lower than the patency rate after vasovasostomy and the pregnancy rate is time dependent. In this study, we evaluated the influence of reproductive tract obstruction on expression of epididymal proteins and their restoration after patency. Adult male Wistar rats were studied 30, 60 and 120 days after vasectomy, 30 days after vasovasostomy or after sham operations. Two-dimensional gel electrophoresis, mass-spectrometric technique, multidatabase search, Western blotting and real-time PCR were used to analyze the expression regulation of epididymal proteins. Total integrated intensity and total spot area of autoradiograms showed a consistent downward trend with time after obstruction, and this trend remained after patency. The intensity of the autoradiographic spots in three patency groups showed three trends: a downward trend, similar intensity and an upward trend compared with the correspondent obstruction group, respectively. Further verified experiments on human epididymis 2 (HE2), fertilization antigen-1 (FA-1), clusterin and PH20 demonstrated that compared with the correspondent obstruction group, the translation levels of HE2 and the mRNA transcription levels of HE2 showed an upward trend in patency groups, especially in the groups of obstruction for 60 days where the expression levels of HE2 were significantly upregulated after patency (P<0.05). Reproductive tract obstruction provokes a disregulation of gene expression in the epididymis and this disregulation remained after patency. Successful reversal may recover some proteins and the recovery is time dependent. Obstruction differentially alters mRNA transcription of different proteins and the content of proteins seemed to be easier to be influenced than the gene transcription.

Estimation of salivary flow rate, pH, buffer capacity, calcium, total protein content and total antioxidant capacity in relation to dental caries severity, age and gender.

PubMed

Pandey, Pallavi; Reddy, N Venugopal; Rao, V Arun Prasad; Saxena, Aditya; Chaudhary, C P

2015-03-01

The aim of the study was to evaluate salivary flow rate, pH, buffering capacity, calcium, total protein content and total antioxidant capacity in relation to dental caries, age and gender. The study population consisted of 120 healthy children aged 7-15 years that was further divided into two groups: 7-10 years and 11-15 years. In this 60 children with DMFS/dfs = 0 and 60 children with DMFS/dfs ≥5 were included. The subjects were divided into two groups; Group A: Children with DMFS/dfs = 0 (caries-free) Group B: Children with DMFS/dfs ≥5 (caries active). Unstimulated saliva samples were collected from all groups. Flow rates were determined, and samples analyzed for pH, buffer capacity, calcium, total protein and total antioxidant status. Salivary antioxidant activity is measured with spectrophotometer by an adaptation of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonate) assays. The mean difference of the two groups; caries-free and caries active were proved to be statistically significant (P < 0.05) for salivary calcium, total protein and total antioxidant level for both the sexes in the age group 7-10 years and for the age 11-15 years the mean difference of the two groups were proved to be statistically significant (P < 0.05) for salivary calcium level for both the sexes. Salivary total protein and total antioxidant level were proved to be statistically significant for male children only. In general, total protein and total antioxidants in saliva were increased with caries activity. Calcium content of saliva was found to be more in caries-free group and increased with age.

The 15N-leucine single-injection method allows for determining endogenous losses and true digestibility of amino acids in cecectomized roosters.

PubMed

Hu, Rujiu; Li, Jing; Soomro, Rab Nawaz; Wang, Fei; Feng, Yan; Yang, Xiaojun; Yao, Junhu

2017-01-01

This study was conducted to assess the influence of dietary protein content in poultry when using the 15N-leucine single-injection method to determine endogenous amino acid losses (EAALs) in poultry. Forty-eight cecectomized roosters (2.39 ± 0.23 kg) were randomly allocated to eight dietary treatments containing protein levels of 0, 3%, 6%, 9%, 12%, 15%, 18% and 21%. Each bird was precisely fed an experimental diet of 25 g/kg of body weight. After feeding, all roosters were subcutaneously injected with a 15N-leucine solution at a dose of 20 mg/kg of body weight. Blood was sampled 23 h after the injection, and excreta samples were continuously collected during the course of the 48-h experiment. The ratio of 15N-enrichment of leucine in crude mucin to free leucine in plasma ranged from 0.664 to 0.763 and remained relatively consistent (P > 0.05) across all treatments. The amino acid (AA) profiles of total endogenous AAs, except isoleucine, alanine, aspartic acid, cysteine, proline and serine, were not influenced (P > 0.05) by dietary protein contents. The predominant endogenous AAs in the excreta were glutamic acid, aspartic acid, threonine, serine and proline. The order of the relative proportions of these predominant AAs also remained relatively constant (P > 0.05). The endogenous losses of total AAs determined with the 15N-leucine single-injection method increased curvilinearly with the dietary protein contents. The true digestibility of most AAs and total AAs was independent of their respective dietary protein levels. Collectively, the 15N-leucine single-injection method is appropriate for determining EAALs and the true digestibility of AAs in poultry fed varying levels of protein-containing ingredients.

The 15N-leucine single-injection method allows for determining endogenous losses and true digestibility of amino acids in cecectomized roosters

PubMed Central

Hu, Rujiu; Li, Jing; Soomro, Rab Nawaz; Wang, Fei; Feng, Yan; Yang, Xiaojun

2017-01-01

This study was conducted to assess the influence of dietary protein content in poultry when using the 15N-leucine single-injection method to determine endogenous amino acid losses (EAALs) in poultry. Forty-eight cecectomized roosters (2.39 ± 0.23 kg) were randomly allocated to eight dietary treatments containing protein levels of 0, 3%, 6%, 9%, 12%, 15%, 18% and 21%. Each bird was precisely fed an experimental diet of 25 g/kg of body weight. After feeding, all roosters were subcutaneously injected with a 15N-leucine solution at a dose of 20 mg/kg of body weight. Blood was sampled 23 h after the injection, and excreta samples were continuously collected during the course of the 48-h experiment. The ratio of 15N-enrichment of leucine in crude mucin to free leucine in plasma ranged from 0.664 to 0.763 and remained relatively consistent (P > 0.05) across all treatments. The amino acid (AA) profiles of total endogenous AAs, except isoleucine, alanine, aspartic acid, cysteine, proline and serine, were not influenced (P > 0.05) by dietary protein contents. The predominant endogenous AAs in the excreta were glutamic acid, aspartic acid, threonine, serine and proline. The order of the relative proportions of these predominant AAs also remained relatively constant (P > 0.05). The endogenous losses of total AAs determined with the 15N-leucine single-injection method increased curvilinearly with the dietary protein contents. The true digestibility of most AAs and total AAs was independent of their respective dietary protein levels. Collectively, the 15N-leucine single-injection method is appropriate for determining EAALs and the true digestibility of AAs in poultry fed varying levels of protein-containing ingredients. PMID:29166671

Brassica rapa Has Three Genes That Encode Proteins Associated with Different Neutral Lipids in Plastids of Specific Tissues1

PubMed Central

Kim, Hyun Uk; Wu, Sherry S.H.; Ratnayake, Chandra; Huang, Anthony H.C.

2001-01-01

Plastid lipid-associated protein (PAP), a predominant structural protein associated with carotenoids and other non-green neutral lipids in plastids, was shown to be encoded by a single nuclear gene in several species. Here we report three PAP genes in the diploid Brassica rapa; the three PAPs are associated with different lipids in specific tissues. Pap1 and Pap2 are more similar to each other (84% amino acid sequence identity) than to Pap3 (46% and 44%, respectively) in the encoded mature proteins. Pap1 transcript was most abundant in the maturing anthers (tapetum) and in lesser amounts in leaves, fruit coats, seeds, and sepals; Pap2 transcript was abundant only in the petals; and Pap3 transcript had a wide distribution, but at minimal levels in numerous organs. Immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that most organs had several nanograms of PAP1 or PAP2 per milligram of total protein, the highest amounts being in the anthers (10.9 μg mg−1 PAP1) and petals (6.6 μg mg−1 PAP2), and that they had much less PAP3 (<0.02 μg mg−1). In these organs PAP was localized in isolated plastid fractions. Plants were subjected to abiotic stresses; drought and ozone reduced the levels of the three Pap transcripts, whereas mechanical wounding and altering the light intensity enhanced their levels. We conclude that the PAP gene family consists of several members whose proteins are associated with different lipids and whose expressions are controlled by distinct mechanisms. Earlier reports of the expression of one Pap gene in various organs in a species need to be re-examined. PMID:11351096

The receptor protein tyrosine phosphatase PTPRJ negatively modulates the CD98hc oncoprotein in lung cancer cells

PubMed Central

D’Agostino, Sabrina; Lanzillotta, Delia; Varano, Mariaconcetta; Botta, Cirino; Baldrini, Antonio; Bilotta, Anna; Scalise, Stefania; Dattilo, Vincenzo; Amato, Rosario; Gaudio, Eugenio; Paduano, Francesco; Palmieri, Camillo; Iuliano, Rodolfo; Perrotti, Nicola; Indiveri, Cesare; Fusco, Alfredo; Gaspari, Marco; Trapasso, Francesco

2018-01-01

PTPRJ, a receptor protein tyrosine phosphatase strongly downregulated in human cancer, displays tumor suppressor activity by negatively modulating several proteins involved in proliferating signals. Here, through a proteomic-based approach, we identified a list of potential PTPRJ-interacting proteins and among them we focused on CD98hc, a type II glycosylated integral membrane protein encoded by SLC3A2, corresponding to the heavy chain of a heterodimeric transmembrane amino-acid transporter, including LAT1. CD98hc is widely overexpressed in several types of cancers and contributes to the process of tumorigenesis by interfering with cell proliferation, adhesion, and migration. We first validated PTPRJ-CD98hc interaction, then demonstrated that PTPRJ overexpression dramatically reduces CD98hc protein levels in A549 lung cancer cells. In addition, following to the treatment of PTPRJ-transduced cells with MG132, a proteasome inhibitor, CD98hc levels did not decrease compared to controls, indicating that PTPRJ is involved in the regulation of CD98hc proteasomal degradation. Moreover, PTPRJ overexpression combined with CD98hc silencing consistently reduced cell proliferation and triggered apoptosis of lung cancer cells. Interestingly, by interrogating the can Evolve database, we observed an inverse correlation between PTPRJ and SLC3A2 gene expression. Indeed, the non-small cell lung cancers (NSCLCs) of patients showing a short survival rate express the lowest and the highest levels of PTPRJ and SLC3A2, respectively. Therefore, the results reported here contribute to shed lights on PTPRJ signaling in cancer cells: moreover, our findings also support the development of a novel anticancer therapeutic approach by targeting the pathway of PTPRJ that is usually downregulated in highly malignant human neoplasias.

Dysregulation of hepatic zinc transporters in a mouse model of alcoholic liver disease

PubMed Central

Sun, Qian; Li, Qiong; Zhong, Wei; Zhang, Jiayang; Sun, Xiuhua; Tan, Xiaobing; Yin, Xinmin; Sun, Xinguo; Zhang, Xiang

2014-01-01

Zinc deficiency is a consistent phenomenon observed in patients with alcoholic liver disease, but the mechanisms have not been well defined. The objective of this study was to determine if alcohol alters hepatic zinc transporters in association with reduction of hepatic zinc levels and if oxidative stress mediates the alterations of zinc transporters. C57BL/6 mice were pair-fed with the Lieber-DeCarli control or ethanol diets for 2, 4, or 8 wk. Chronic alcohol exposure reduced hepatic zinc levels, but increased plasma and urine zinc levels, at all time points. Hepatic zinc finger proteins, peroxisome proliferator-activated receptor-α (PPAR-α) and hepatocyte nuclear factor 4α (HNF-4α), were downregulated in ethanol-fed mice. Four hepatic zinc transporter proteins showed significant alterations in ethanol-fed mice compared with the controls. ZIP5 and ZIP14 proteins were downregulated, while ZIP7 and ZnT7 proteins were upregulated, by ethanol exposure at all time points. Immunohistochemical staining demonstrated that chronic ethanol exposure upregulated cytochrome P-450 2E1 and caused 4-hydroxynonenal accumulation in the liver. For the in vitro study, murine FL-83B hepatocytes were treated with 5 μM 4-hydroxynonenal or 100 μM hydrogen peroxide for 72 h. The results from in vitro studies demonstrated that 4-hydroxynonenal treatment altered ZIP5 and ZIP7 protein abundance, and hydrogen peroxide treatment changed ZIP7, ZIP14, and ZnT7 protein abundance. These results suggest that chronic ethanol exposure alters hepatic zinc transporters via oxidative stress, which might account for ethanol-induced hepatic zinc deficiency. PMID:24924749

Upregulation of capacity for glutathione synthesis in response to amino acid deprivation: regulation of glutamate-cysteine ligase subunits

PubMed Central

Sikalidis, Angelos K.; Mazor, Kevin M.; Lee, Jeong-In; Roman, Heather B.; Hirschberger, Lawrence L.; Stipanuk, Martha H.

2014-01-01

Using HepG2/C3A cells and MEFs, we investigated whether induction of GSH synthesis in response to sulfur amino acid deficiency is mediated by the decrease in cysteine levels or whether it requires a decrease in GSH levels per se. Both the glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunit mRNA levels were upregulated in response to a lack of cysteine or other essential amino acids, independent of GSH levels. This upregulation did not occur in MEFs lacking GCN2 (general control non-derepressible 2, also known as eIF2α kinase 4) or in cells expressing mutant eIF2α lacking the eIF2α kinase Ser51 phosphorylation site, indicating that expression of both GCLC and GCLM was mediated by the GCN2/ATF4 stress response pathway. Only the increase in GCLM mRNA level, however, was accompanied by a parallel increase in protein expression, suggesting that the enhanced capacity for GSH synthesis depended largely on increased association of GCLC with its regulatory subunit. Upregulation of both GCLC and GLCM mRNA levels in response to cysteine deprivation was dependent on new protein synthesis, which is consistent with expression of GCLC and GCLM being mediated by proteins whose synthesis depends on activation of the GCN2/ATF4 pathway. Our data suggest that the regulation of GCLC expression may be mediated by changes in the abundance of transcriptional regulators, whereas the regulation of GCLM expression may be mediated by changes in the abundance of mRNA stabilizing or destabilizing proteins. Upregulation of GCLM levels in response to low cysteine levels may serve to protect the cell in the face of a future stress requiring GSH as an antioxidant or conjugating/detoxifying agent. PMID:24557597

Brain-Derived Neurotrophic Factor (BDNF) protein levels in anxiety disorders: systematic review and meta-regression analysis

PubMed Central

Suliman, Sharain; Hemmings, Sian M. J.; Seedat, Soraya

2013-01-01

Background: Brain-Derived Neurotrophic Factor (BDNF) is a neurotrophin that is involved in the synaptic plasticity and survival of neurons. BDNF is believed to be involved in the pathogenesis of several neuropsychiatric disorders. As findings of BDNF levels in anxiety disorders have been inconsistent, we undertook to conduct a systematic review and meta-analysis of studies that assessed BDNF protein levels in these disorders. Methods: We conducted the review using electronic databases and searched reference lists of relevant articles for any further studies. Studies that measured BDNF protein levels in any anxiety disorder and compared these to a control group were included. Effect sizes of the differences in BDNF levels between anxiety disorder and control groups were calculated. Results: Eight studies with a total of 1179 participants were included. Initial findings suggested that BDNF levels were lower in individuals with any anxiety disorder compared to those without [Standard Mean Difference (SMD) = −0.94 (−1.75, −0.12), p ≤ 0.05]. This was, however, dependent on source of BDNF protein [plasma: SMD = −1.31 (−1.69, −0.92), p ≤ 0.01; serum: SMD = −1.06 (−2.27, 0.16), p ≥ 0.01] and type of anxiety disorder [PTSD: SMD = −0.05 (−1.66, 1.75), p ≥ 0.01; OCD: SMD = −2.33 (−4.21, −0.45), p ≤ 0.01]. Conclusion: Although BDNF levels appear to be reduced in individuals with an anxiety disorder, this is not consistent across the various anxiety disorders and may largely be explained by the significantly lowered BDNF levels found in OCD. Results further appear to be mediated by differences in sampling methods. Findings are, however, limited by the lack of research in this area, and given the potential for BDNF as a biomarker of anxiety disorders, it would be useful to clarify the relationship further. PMID:23908608

Growth-Phase-Specific Modulation of Cell Morphology and Gene Expression by an Archaeal Histone Protein.

PubMed

Dulmage, Keely A; Todor, Horia; Schmid, Amy K

2015-09-08

In all three domains of life, organisms use nonspecific DNA-binding proteins to compact and organize the genome as well as to regulate transcription on a global scale. Histone is the primary eukaryotic nucleoprotein, and its evolutionary roots can be traced to the archaea. However, not all archaea use this protein as the primary DNA-packaging component, raising questions regarding the role of histones in archaeal chromatin function. Here, quantitative phenotyping, transcriptomic, and proteomic assays were performed on deletion and overexpression mutants of the sole histone protein of the hypersaline-adapted haloarchaeal model organism Halobacterium salinarum. This protein is highly conserved among all sequenced haloarchaeal species and maintains hallmark residues required for eukaryotic histone functions. Surprisingly, despite this conservation at the sequence level, unlike in other archaea or eukaryotes, H. salinarum histone is required to regulate cell shape but is not necessary for survival. Genome-wide expression changes in histone deletion strains were global, significant but subtle in terms of fold change, bidirectional, and growth phase dependent. Mass spectrometric proteomic identification of proteins from chromatin enrichments yielded levels of histone and putative nucleoid-associated proteins similar to those of transcription factors, consistent with an open and transcriptionally active genome. Taken together, these data suggest that histone in H. salinarum plays a minor role in DNA compaction but important roles in growth-phase-dependent gene expression and regulation of cell shape. Histone function in haloarchaea more closely resembles a regulator of gene expression than a chromatin-organizing protein like canonical eukaryotic histone. Histones comprise the major protein component of eukaryotic chromatin and are required for both genome packaging and global regulation of expression. The current paradigm maintains that archaea whose genes encode histone also use these proteins to package DNA. In contrast, here we demonstrate that the sole histone encoded in the genome of the salt-adapted archaeon Halobacterium salinarum is both unessential and unlikely to be involved in DNA compaction despite conservation of residues important for eukaryotic histones. Rather, H. salinarum histone is required for global regulation of gene expression and cell shape. These data are consistent with the hypothesis that H. salinarum histone, strongly conserved across all other known salt-adapted archaea, serves a novel role in gene regulation and cell shape maintenance. Given that archaea possess the ancestral form of eukaryotic histone, this study has important implications for understanding the evolution of histone function. Copyright © 2015 Dulmage et al.

Conformational co-dependence between Plasmodium berghei LCCL proteins promotes complex formation and stability.

PubMed

Saeed, Sadia; Tremp, Annie Z; Dessens, Johannes T

2012-10-01

Malaria parasites express a conserved family of LCCL-lectin adhesive-like domain proteins (LAPs) that have essential functions in sporozoite transmission. In Plasmodium falciparum all six family members are expressed in gametocytes and form a multi-protein complex. Intriguingly, knockout of P. falciparum LCCL proteins adversely affects expression of other family members at protein, but not at mRNA level, a phenomenon termed co-dependent expression. Here, we investigate this in Plasmodium berghei by crossing a PbLAP1 null mutant parasite with a parasite line expressing GFP-tagged PbLAP3 that displays strong fluorescence in gametocytes. Selected and validated double mutants show normal synthesis and subcellular localization of PbLAP3::GFP. However, GFP-based fluorescence is dramatically reduced without PbLAP1 present, indicating that PbLAP1 and PbLAP3 interact. Moreover, absence of PbLAP1 markedly reduces the half-life of PbLAP3, consistent with a scenario of misfolding. These findings unveil a potential mechanism of conformational interdependence that facilitates assembly and stability of the functional LCCL protein complex. Copyright © 2012 Elsevier B.V. All rights reserved.

Systemic delivery of factor IX messenger RNA for protein replacement therapy

PubMed Central

Ramaswamy, Suvasini; Tonnu, Nina; Tachikawa, Kiyoshi; Limphong, Pattraranee; Vega, Jerel B.; Karmali, Priya P.; Chivukula, Pad; Verma, Inder M.

2017-01-01

Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4–6 h) that remains stable for up to 4–6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA–LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable. PMID:28202722

External Quality Control for Dried Blood Spot Based C-reactive Protein Assay: Experience from the Indonesia Family Life Survey and the Longitudinal Aging Study in India

PubMed Central

Hu, Peifeng; Herningtyas, Elizabeth H.; Kale, Varsha; Crimmins, Eileen M.; Risbud, Arun R.; McCreath, Heather; Lee, Jinkook; Strauss, John; O’Brien, Jennifer C.; Bloom, David E.; Seeman, Teresa E.

2015-01-01

Measurement of C-reactive protein, a marker of inflammation, in dried blood spots has been increasingly incorporated in community-based social surveys internationally. Although the dried blood spot based CRP assay protocol has been validated in the United States, it remains unclear whether laboratories in other less developed countries can generate C-reactive protein results of similar quality. We therefore conducted external quality monitoring for dried blood spot based C-reactive protein measurement for the Indonesia Family Life Survey and the Longitudinal Aging Study in India. Our results show that dried blood spot based C-reactive protein results in these two countries have excellent and consistent correlations with serum-based values and dried blood spot based results from the reference laboratory in the United States. Even though the results from duplicate samples may have fluctuations in absolute values over time, the relative order of C-reactive protein levels remains similar and the estimates are reasonably precise for population-based studies that investigate the association between socioeconomic factors and health. PMID:25879265

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

NASA Astrophysics Data System (ADS)

Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

2017-01-01

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

Effect of gluten, egg and soy proteins on the rheological and thermo-mechanical properties of wholegrain rice flour.

PubMed

Pătraşcu, Livia; Banu, Iuliana; Vasilean, Ina; Aprodu, Iuliana

2017-03-01

The effect of protein addition on the rheological, thermo-mechanical and baking properties of wholegrain rice flour was investigated. Gluten, powdered eggs and soy protein concentrate were first analyzed in terms of rheological properties, alone and in admixture with rice flour. The temperature ramp tests showed clear differences in the rheological behavior of the batters supplemented with different proteins. The highest thermal stability was observed in case of soy protein samples. Frequency sweep tests indicated significant improvements of the rheological properties of rice flour supplemented with 15% gluten or soy proteins. The thermo-mechanical tests showed that, due to the high fat contents and low level of free water, the dough samples containing powdered eggs exhibited the highest stability. Addition of gluten resulted in a significant decrease of the dough development time, whereas samples with powdered eggs and soy proteins were more difficult to hydrate. The incorporation of proteins into the rice flour-based dough formulations significantly affected starch behavior by decreasing the peak consistency values. Concerning the quality of the rice flour-based breads, soy protein addition resulted in lighter crumb color and increased texture attributes, samples with gluten had better resilience and adhesiveness, whereas breads with egg protein were less brittle.

N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae.

PubMed

Ojima-Kato, Teruyo; Nagai, Satomi; Nakano, Hideo

2017-05-01

Despite advances in microbial protein expression systems, low production of proteins remains a great concern for some genes. Here we report that the insertion of a short peptide tag, consisting of Ser-Lys-Ile-Lys (SKIK), adjacent to the start codon of genes encoding difficult-to-express proteins can increase protein expression in Escherichia coli and Saccharomyces cerevisiae. Protein expression levels of a mouse monoclonal antibody (mAb), rabbit mAbs obtained from clonal B cells, and an artificially designed peptide were significantly increased simply by the addition of the SKIK tag in E. coli systems. In particular, a ∼30-fold increase in protein production was observed for the mouse mAb, and the artificially designed peptide band became detectable in sodium dodecyl sulfate-poly acrylamide gel electrophoresis after coomassie brilliant blue staining or western blotting on adding the SKIK tag. The tag also increased the expression of tagged proteins in S. cerevisiae and an E. coli cell-free protein synthesis system. Although the mechanism of high protein expression on addition of the tag is unclear, our findings offer great benefits to biotechnology research and industry. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The effect of different protein hydrolysate/carbohydrate mixtures on postprandial glucagon and insulin responses in healthy subjects.

PubMed

Claessens, M; Calame, W; Siemensma, A D; van Baak, M A; Saris, W H M

2009-01-01

To study the effect of four protein hydrolysates from vegetable (pea, gluten, rice and soy) and two protein hydrolysates from animal origin (whey and egg) on glucagon and insulin responses. Eight healthy normal-weight male subjects participated in this study. The study employed a repeated-measures design with Latin square randomization and single-blind trials. Protein hydrolysates used in this study (pea, rice, soy, gluten, whey and egg protein hydrolysate) consisted of 0.2 g hydrolysate per kg body weight (bw) and 0.2 g maltodextrin per kg bw and were compared to maltodextrin alone. Postprandial plasma glucose, glucagon, insulin and amino acids were determined over 2 h. All protein hydrolysates induced an enhanced insulin secretion compared to maltodextrin alone and a correspondingly low plasma glucose response. A significant difference was observed in area under the curve (AUC) for plasma glucagon between protein hydrolysates and the maltodextrin control drink (P<0.05). Gluten protein hydrolysate induced the lowest glucagon response. High amino-acid-induced glucagon response does not necessarily go together with low insulin response. Protein hydrolysate source affects AUC for glucagon more profoundly than for insulin, although the protein load used in this study seemed to be at lower level for significant physiological effects.

Proteomic analysis of PSD-93 knockout mice following the induction of ischemic cerebral injury.

PubMed

Rong, Rong; Yang, Hui; Rong, Liangqun; Wei, Xiue; Li, Qingjie; Liu, Xiaomei; Gao, Hong; Xu, Yun; Zhang, Qingxiu

2016-03-01

Postsynaptic density protein-93 (PSD-93) is enriched in the postsynaptic density and is involved in N-methyl-d-aspartate receptor (NMDAR) triggered neurotoxicity through PSD-93/NMDAR/nNOS signaling pathway. In the present study, we found that PSD-93 deficiency reduced infarcted volume and neurological deficits induced by transient middle cerebral artery occlusion (tMCAO) in the mice. To identify novel targets of PSD-93 related neurotoxicity, we applied isobaric tags for relative and absolute quantitative (iTRAQ) labeling and combined this labeling with on-line two-dimensional LC/MS/MS technology to elucidate the changes in protein expression in PSD-93 knockout mice following tMCAO. The proteomic data set consisted of 1892 proteins. Compared to control group, differences in expression levels in ischemic group >1.5-fold and <0.66-fold were considered as differential expression. A total of 104 unique proteins with differential abundance levels were identified, among which 17 proteins were selected for further validation. Gene ontology analysis using UniProt database revealed that these differentially expressed proteins are involved in diverse function such as synaptic transmission, neuronal neurotransmitter and ion transport, modification of organelle membrane components. Moreover, network analysis revealed that the interacting proteins were involved in the transport of synaptic vesicles, the integrity of synaptic membranes and the activation of the ionotropic glutamate receptors NMDAR1 and NMDAR2B. Finally, RT-PCR and Western blot analysis showed that SynGAP, syntaxin-1A, protein kinase C β, and voltage-dependent L-type calcium channels were inhibited by ischemia-reperfusion. Identification of these proteins provides valuable clues to elucidate the mechanisms underlying the actions of PSD-93 in ischemia-reperfusion induced neurotoxicity. Copyright © 2015 Elsevier Inc. All rights reserved.

Quality by design approach for viral clearance by protein a chromatography

PubMed Central

Zhang, Min; Miesegaes, George R; Lee, Michael; Coleman, Daniel; Yang, Bin; Trexler-Schmidt, Melody; Norling, Lenore; Lester, Philip; Brorson, Kurt A; Chen, Qi

2014-01-01

Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus-like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X-MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design-of-experiment (DoE)—type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product. PMID:23860745

Characterization of the sex-dependent myocardial S-nitrosothiol proteome

PubMed Central

Shao, Qin; Fallica, Jonathan; Casin, Kevin M.; Murphy, Elizabeth; Steenbergen, Charles

2015-01-01

Premenopausal women exhibit endogenous cardioprotective signaling mechanisms that are thought to result from the beneficial effects of estrogen, which we have shown to increase protein S-nitrosylation in the heart. S-nitrosylation is a labile protein modification that increases with a number of different forms of cardioprotection, including ischemic preconditioning. Herein, we sought to identify a potential role for protein S-nitrosylation in sex-dependent cardioprotection. We utilized a Langendorff-perfused mouse heart model of ischemia-reperfusion injury with male and female hearts, and S-nitrosylation-resin-assisted capture with liquid chromatography tandem mass spectrometry to identify S-nitrosylated proteins and modification sites. Consistent with previous studies, female hearts exhibited resilience to injury with a significant increase in functional recovery compared with male hearts. In a separate set of hearts, we identified a total of 177 S-nitrosylated proteins in female hearts at baseline compared with 109 S-nitrosylated proteins in male hearts. Unique S-nitrosylated proteins in the female group included the F1FO-ATPase and cyclophilin D. We also utilized label-free peptide analysis to quantify levels of common S-nitrosylated identifications and noted that the S-nitrosylation of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a was nearly 70% lower in male hearts compared with female, with no difference in expression. Furthermore, we found a significant increase in endothelial nitric oxide synthase expression, phosphorylation, and total nitric oxide production in female hearts compared with males, likely accounting for the enhanced S-nitrosylation protein levels in female hearts. In conclusion, we identified a number of novel S-nitrosylated proteins in female hearts that are likely to contribute to sex-dependent cardioprotection. PMID:26702143

Isolation and Proteomic Characterization of the Arabidopsis Golgi Defines Functional and Novel Components Involved in Plant Cell Wall Biosynthesis1[W][OA

PubMed Central

Parsons, Harriet T.; Christiansen, Katy; Knierim, Bernhard; Carroll, Andrew; Ito, Jun; Batth, Tanveer S.; Smith-Moritz, Andreia M.; Morrison, Stephanie; McInerney, Peter; Hadi, Masood Z.; Auer, Manfred; Mukhopadhyay, Aindrila; Petzold, Christopher J.; Scheller, Henrik V.; Loqué, Dominique; Heazlewood, Joshua L.

2012-01-01

The plant Golgi plays a pivotal role in the biosynthesis of cell wall matrix polysaccharides, protein glycosylation, and vesicle trafficking. Golgi-localized proteins have become prospective targets for reengineering cell wall biosynthetic pathways for the efficient production of biofuels from plant cell walls. However, proteomic characterization of the Golgi has so far been limited, owing to the technical challenges inherent in Golgi purification. In this study, a combination of density centrifugation and surface charge separation techniques have allowed the reproducible isolation of Golgi membranes from Arabidopsis (Arabidopsis thaliana) at sufficiently high purity levels for in-depth proteomic analysis. Quantitative proteomic analysis, immunoblotting, enzyme activity assays, and electron microscopy all confirm high purity levels. A composition analysis indicated that approximately 19% of proteins were likely derived from contaminating compartments and ribosomes. The localization of 13 newly assigned proteins to the Golgi using transient fluorescent markers further validated the proteome. A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome, marking an appreciable advancement in numbers of Golgi-localized proteins. A significant proportion of proteins likely involved in matrix polysaccharide biosynthesis were identified. The potential within this proteome for advances in understanding Golgi processes has been demonstrated by the identification and functional characterization of the first plant Golgi-resident nucleoside diphosphatase, using a yeast complementation assay. Overall, these data show key proteins involved in primary cell wall synthesis and include a mixture of well-characterized and unknown proteins whose biological roles and importance as targets for future research can now be realized. PMID:22430844

HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells

PubMed Central

Lemay, Julie; Maidou-Peindara, Priscilla; Bader, Thomas; Ennifar, Eric; Rain, Jean-Christophe; Benarous, Richard; Liu, Lang Xia

2008-01-01

Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex – the reverse transcription complex (RTC) – consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein – the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT. PMID:18544151

Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein.

PubMed

Ridker, Paul M; Danielson, Eleanor; Fonseca, Francisco A H; Genest, Jacques; Gotto, Antonio M; Kastelein, John J P; Koenig, Wolfgang; Libby, Peter; Lorenzatti, Alberto J; MacFadyen, Jean G; Nordestgaard, Børge G; Shepherd, James; Willerson, James T; Glynn, Robert J

2008-11-20

Increased levels of the inflammatory biomarker high-sensitivity C-reactive protein predict cardiovascular events. Since statins lower levels of high-sensitivity C-reactive protein as well as cholesterol, we hypothesized that people with elevated high-sensitivity C-reactive protein levels but without hyperlipidemia might benefit from statin treatment. We randomly assigned 17,802 apparently healthy men and women with low-density lipoprotein (LDL) cholesterol levels of less than 130 mg per deciliter (3.4 mmol per liter) and high-sensitivity C-reactive protein levels of 2.0 mg per liter or higher to rosuvastatin, 20 mg daily, or placebo and followed them for the occurrence of the combined primary end point of myocardial infarction, stroke, arterial revascularization, hospitalization for unstable angina, or death from cardiovascular causes. The trial was stopped after a median follow-up of 1.9 years (maximum, 5.0). Rosuvastatin reduced LDL cholesterol levels by 50% and high-sensitivity C-reactive protein levels by 37%. The rates of the primary end point were 0.77 and 1.36 per 100 person-years of follow-up in the rosuvastatin and placebo groups, respectively (hazard ratio for rosuvastatin, 0.56; 95% confidence interval [CI], 0.46 to 0.69; P<0.00001), with corresponding rates of 0.17 and 0.37 for myocardial infarction (hazard ratio, 0.46; 95% CI, 0.30 to 0.70; P=0.0002), 0.18 and 0.34 for stroke (hazard ratio, 0.52; 95% CI, 0.34 to 0.79; P=0.002), 0.41 and 0.77 for revascularization or unstable angina (hazard ratio, 0.53; 95% CI, 0.40 to 0.70; P<0.00001), 0.45 and 0.85 for the combined end point of myocardial infarction, stroke, or death from cardiovascular causes (hazard ratio, 0.53; 95% CI, 0.40 to 0.69; P<0.00001), and 1.00 and 1.25 for death from any cause (hazard ratio, 0.80; 95% CI, 0.67 to 0.97; P=0.02). Consistent effects were observed in all subgroups evaluated. The rosuvastatin group did not have a significant increase in myopathy or cancer but did have a higher incidence of physician-reported diabetes. In this trial of apparently healthy persons without hyperlipidemia but with elevated high-sensitivity C-reactive protein levels, rosuvastatin significantly reduced the incidence of major cardiovascular events. (ClinicalTrials.gov number, NCT00239681.) 2008 Massachusetts Medical Society

M-Finder: Uncovering functionally associated proteins from interactome data integrated with GO annotations

PubMed Central

2013-01-01

Background Protein-protein interactions (PPIs) play a key role in understanding the mechanisms of cellular processes. The availability of interactome data has catalyzed the development of computational approaches to elucidate functional behaviors of proteins on a system level. Gene Ontology (GO) and its annotations are a significant resource for functional characterization of proteins. Because of wide coverage, GO data have often been adopted as a benchmark for protein function prediction on the genomic scale. Results We propose a computational approach, called M-Finder, for functional association pattern mining. This method employs semantic analytics to integrate the genome-wide PPIs with GO data. We also introduce an interactive web application tool that visualizes a functional association network linked to a protein specified by a user. The proposed approach comprises two major components. First, the PPIs that have been generated by high-throughput methods are weighted in terms of their functional consistency using GO and its annotations. We assess two advanced semantic similarity metrics which quantify the functional association level of each interacting protein pair. We demonstrate that these measures outperform the other existing methods by evaluating their agreement to other biological features, such as sequence similarity, the presence of common Pfam domains, and core PPIs. Second, the information flow-based algorithm is employed to discover a set of proteins functionally associated with the protein in a query and their links efficiently. This algorithm reconstructs a functional association network of the query protein. The output network size can be flexibly determined by parameters. Conclusions M-Finder provides a useful framework to investigate functional association patterns with any protein. This software will also allow users to perform further systematic analysis of a set of proteins for any specific function. It is available online at http://bionet.ecs.baylor.edu/mfinder PMID:24565382

Protein structure and evolution: are they constrained globally by a principle derived from information theory?

PubMed

Hatton, Leslie; Warr, Gregory

2015-01-01

That the physicochemical properties of amino acids constrain the structure, function and evolution of proteins is not in doubt. However, principles derived from information theory may also set bounds on the structure (and thus also the evolution) of proteins. Here we analyze the global properties of the full set of proteins in release 13-11 of the SwissProt database, showing by experimental test of predictions from information theory that their collective structure exhibits properties that are consistent with their being guided by a conservation principle. This principle (Conservation of Information) defines the global properties of systems composed of discrete components each of which is in turn assembled from discrete smaller pieces. In the system of proteins, each protein is a component, and each protein is assembled from amino acids. Central to this principle is the inter-relationship of the unique amino acid count and total length of a protein and its implications for both average protein length and occurrence of proteins with specific unique amino acid counts. The unique amino acid count is simply the number of distinct amino acids (including those that are post-translationally modified) that occur in a protein, and is independent of the number of times that the particular amino acid occurs in the sequence. Conservation of Information does not operate at the local level (it is independent of the physicochemical properties of the amino acids) where the influences of natural selection are manifest in the variety of protein structure and function that is well understood. Rather, this analysis implies that Conservation of Information would define the global bounds within which the whole system of proteins is constrained; thus it appears to be acting to constrain evolution at a level different from natural selection, a conclusion that appears counter-intuitive but is supported by the studies described herein.

Oxidative stress biomarkers in two resident species, mullet (Mugil cephalus) and flounder (Platichthys flesus), from a polluted site in River Douro Estuary, Portugal.

PubMed

Ferreira, M; Moradas-Ferreira, P; Reis-Henriques, M A

2005-01-18

Exposure of marine animals to certain pollutants can enhance reactive oxygen species (ROS) production with subsequent damage to macromolecules and alterations in oxidant defences levels. Aimed at correlating the tissue concentration of certain contaminants (PCBs, DDT) with antioxidant defence levels and oxidative damages, two fish species with different life strategies (mullet, Mugil cephalus, and flounder, Platichthys flesus) were collected in the Douro Estuary (NW Portugal). After capture, the fish were left to depurate for 1 month in clean seawater. The levels of the two antioxidant enzyme activities revealed that they are species-dependent with mullet's livers showing higher superoxide dismutase (SOD) (13.2+/-0.5 U/mg protein) and catalase (CAT) (15.5+/-1.0 mmol/min/mg protein) activities than flounder (SOD: 7.9+/-0.9 U/mg protein; CAT: 11.1+/-0.8 mmol/min/mg protein). After 1 month in captivity the antioxidant enzymes activities in liver decreased in mullets, while for flounders the responses were not consistent because during the experimental period flounders did not ate and responses of antioxidant enzymes and oxidative damages were dependent on the fasting condition. The liver oxidative damages were evaluated by estimating oxidised lipids and proteins. Both species showed similar levels for these two parameters. The hepatic lipid peroxidation in flounder increased after 1 month in captivity, while in mullet an increase was observed only in summer and autumn. The oxidised protein content increased for both species after the depuration period. This study reveals differences between species under oxidative stress when exposed to pollutants. In a clean environment, the mullet's primary antioxidant defences decreased indicating that the animals living in Douro estuary were facing an oxidative stress. The data indicate that, namely in mullet, the presence of pollutants induce oxidative stress responses.

Down-regulation of Jab1, HIF-1alpha, and VEGF by Moloney murine leukemia virus-ts1 infection: a possible cause of neurodegeneration.

PubMed

Lungu, Gina F; Stoica, George; Wong, Paul K Y

2008-05-01

Moloney murine leukemia virus-temperature sensitive (MoMuLV-ts1)-mediated neuronal death is a result of both loss of glial support and release of cytokines and neurotoxins from ts1-infected glial cells. Here the authors propose vascular endothelial growth factor (VEGF) down-regulation as another contributory factor in neuronal degeneration induced by ts1 infection. To determine how ts1 affects VEGF expression in ts1-infected brain, the authors examined the expression of several proteins that are important in regulating the expression of VEGF. The authors found significant decreases in Jun-activating domain-binding protein 1 (Jab1), hypoxia-inducible factor (HIF)-1alpha, and VEGF levels and increases in p53 protein levels in ts1-infected brains compared to noninfected control brains. The authors suggest that a decrease Jab1 expression in ts1 infection leads to accumulation of p53, which binds to HIF-1alpha to accelerate its degradation. A rapid degradation of HIF-1alpha leads to decreased VEGF production and secretion. Considering that endothelial cells are the most conspicuous in virus replication and production in ts1 infection, but are not killed by the infection, the authors examined the expression of these proteins using infected and noninfected mouse cerebrovascular endothelial (CVE) cells. The ts1- infected CVE cells showed decreased Jab1, HIF-1alpha, and VEGF mRNA and protein levels and increased p53 protein levels compared with noninfected cells, consistent with the results found in vivo. These results confirm that ts1 infection results in insufficient secretion of VEGF from endothelial cells and may result in decreased neuroprotection. This study suggested that ts1-mediated neuropathology in mice may result from changes in expression and activity of Jab1, p53, and HIF-1alpha, with a final target on VEGF expression and neuronal degeneration.

Quantification of chitinase and thaumatin-like proteins in grape juices and wines.

PubMed

Le Bourse, D; Conreux, A; Villaume, S; Lameiras, P; Nuzillard, J-M; Jeandet, P

2011-09-01

Chitinases and thaumatin-like proteins are important grape proteins as they have a great influence on wine quality. The quantification of these proteins in grape juices and wines, along with their purification, is therefore crucial to study their intrinsic characteristics and the exact role they play in wines. The main isoforms of these two proteins from Chardonnay grape juice were thus purified by liquid chromatography. Two fast protein liquid chromatography (FLPC) steps allowed the fractionation and purification of the juice proteins, using cation exchange and hydrophobic interaction media. A further high-performance liquid chromatography (HPLC) step was used to achieve higher purity levels. Fraction assessment was achieved by mass spectrometry. Fraction purity was determined by HPLC to detect the presence of protein contaminants, and by nuclear magnetic resonance (NMR) spectroscopy to detect the presence of organic contaminants. Once pure fractions of lyophilized chitinase and thaumatin-like protein were obtained, ultra-HPLC (UHPLC) and enzyme-linked immunosorbent assay (ELISA) calibration curves were constructed. The quantification of these proteins in different grape juice and wine samples was thus achieved for the first time with both techniques through comparison with the purified protein calibration curve. UHPLC and ELISA showed very consistent results (less than 16% deviation for both proteins) and either could be considered to provide an accurate and reliable quantification of proteins in the oenology field.

Hypothalamic-Pituitary-Adrenal Axis Dysfunction and Illness Progression in Bipolar Disorder

PubMed Central

Vasconcelos-Moreno, Mirela Paiva; Gubert, Carolina; dos Santos, Bárbara Tietböhl Martins Quadros; Sartori, Juliana; Eisele, Bárbara; Ferrari, Pamela; Fijtman, Adam; Rüegg, Joëlle; Gassen, Nils Christian; Kapczinski, Flávio; Rein, Theo; Kauer-Sant’Anna, Márcia

2015-01-01

Background: Impaired stress resilience and a dysfunctional hypothalamic-pituitary-adrenal (HPA) axis are suggested to play key roles in the pathophysiology of illness progression in bipolar disorder (BD), but the mechanisms leading to this dysfunction have never been elucidated. This study aimed to examine HPA axis activity and underlying molecular mechanisms in patients with BD and unaffected siblings of BD patients. Methods: Twenty-four euthymic patients with BD, 18 siblings of BD patients, and 26 healthy controls were recruited for this study. All subjects underwent a dexamethasone suppression test followed by analyses associated with the HPA axis and the glucocorticoid receptor (GR). Results: Patients with BD, particularly those at a late stage of illness, presented increased salivary post-dexamethasone cortisol levels when compared to controls (p = 0.015). Accordingly, these patients presented reduced ex vivo GR responsiveness (p = 0.008) and increased basal protein levels of FK506-binding protein 51 (FKBP51, p = 0.012), a co-chaperone known to desensitize GR, in peripheral blood mononuclear cells. Moreover, BD patients presented increased methylation at the FK506-binding protein 5 (FKBP5) gene. BD siblings presented significantly lower FKBP51 protein levels than BD patients, even though no differences were found in FKBP5 basal mRNA levels. Conclusions: Our data suggest that the epigenetic modulation of the FKBP5 gene, along with increased FKBP51 levels, is associated with the GR hyporesponsiveness seen in BD patients. Our findings are consistent with the notion that unaffected first-degree relatives of BD patients share biological factors that influence the disorder, and that such changes are more pronounced in the late stages of the illness. PMID:25522387

Proteomic analysis of trans-hemispheric motor cortex reorganization following contralateral C7 nerve transfer

PubMed Central

Yuan, Yin; Xu, Xiu-yue; Lao, Jie; Zhao, Xin

2018-01-01

Nerve transfer is the most common treatment for total brachial plexus avulsion injury. After nerve transfer, the movement of the injured limb may be activated by certain movements of the healthy limb at the early stage of recovery, i.e., trans-hemispheric reorganization. Previous studies have focused on functional magnetic resonance imaging and changes in brain-derived neurotrophic factor and growth associated protein 43, but there have been no proteomics studies. In this study, we designed a rat model of total brachial plexus avulsion injury involving contralateral C7 nerve transfer. Isobaric tags for relative and absolute quantitation and western blot assay were then used to screen differentially expressed proteins in bilateral motor cortices. We found that most differentially expressed proteins in both cortices of upper limb were associated with nervous system development and function (including neuron differentiation and development, axonogenesis, and guidance), microtubule and cytoskeleton organization, synapse plasticity, and transmission of nerve impulses. Two key differentially expressed proteins, neurofilament light (NFL) and Thy-1, were identified. In contralateral cortex, the NFL level was upregulated 2 weeks after transfer and downregulated at 1 and 5 months. The Thy-1 level was upregulated from 1 to 5 months. In the affected cortex, the NFL level increased gradually from 1 to 5 months. Western blot results of key differentially expressed proteins were consistent with the proteomic findings. These results indicate that NFL and Thy-1 play an important role in trans-hemispheric organization following total brachial plexus root avulsion and contralateral C7 nerve transfer. PMID:29557385

Overexpression of SERBP1 (Plasminogen activator inhibitor 1 RNA binding protein) in human breast cancer is correlated with favourable prognosis

PubMed Central

2012-01-01

Background Plasminogen activator inhibitor 1 (PAI-1) overexpression is an important prognostic and predictive biomarker in human breast cancer. SERBP1, a protein that is supposed to regulate the stability of PAI-1 mRNA, may play a role in gynaecological cancers as well, since upregulation of SERBP1 was described in ovarian cancer recently. This is the first study to present a systematic characterisation of SERBP1 expression in human breast cancer and normal breast tissue at both the mRNA and the protein level. Methods Using semiquantitative realtime PCR we analysed SERBP1 expression in different normal human tissues (n = 25), and in matched pairs of normal (n = 7) and cancerous breast tissues (n = 7). SERBP1 protein expression was analysed in two independent cohorts on tissue microarrays (TMAs), an initial evaluation set, consisting of 193 breast carcinomas and 48 normal breast tissues, and a second large validation set, consisting of 605 breast carcinomas. In addition, a collection of benign (n = 2) and malignant (n = 6) mammary cell lines as well as breast carcinoma lysates (n = 16) were investigated for SERBP1 expression by Western blot analysis. Furthermore, applying non-radioisotopic in situ hybridisation a subset of normal (n = 10) and cancerous (n = 10) breast tissue specimens from the initial TMA were analysed for SERBP1 mRNA expression. Results SERBP1 is not differentially expressed in breast carcinoma compared to normal breast tissue, both at the RNA and protein level. However, recurrence-free survival analysis showed a significant correlation (P = 0.008) between abundant SERBP1 expression in breast carcinoma and favourable prognosis. Interestingly, overall survival analysis also displayed a tendency (P = 0.09) towards favourable prognosis when SERBP1 was overexpressed in breast cancer. Conclusions The RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a novel breast tumour marker with prognostic significance. Its potential involvement in the plasminogen activator protease cascade warrants further investigation. PMID:23236990

Characterization of the Cardiac Overexpression of HSPB2 Reveals Mitochondrial and Myogenic Roles Supported by a Cardiac HspB2 Interactome.

PubMed

Grose, Julianne H; Langston, Kelsey; Wang, Xiaohui; Squires, Shayne; Mustafi, Soumyajit Banerjee; Hayes, Whitney; Neubert, Jonathan; Fischer, Susan K; Fasano, Matthew; Saunders, Gina Moore; Dai, Qiang; Christians, Elisabeth; Lewandowski, E Douglas; Ping, Peipei; Benjamin, Ivor J

2015-01-01

Small Heat Shock Proteins (sHSPs) are molecular chaperones that transiently interact with other proteins, thereby assisting with quality control of proper protein folding and/or degradation. They are also recruited to protect cells from a variety of stresses in response to extreme heat, heavy metals, and oxidative-reductive stress. Although ten human sHSPs have been identified, their likely diverse biological functions remain an enigma in health and disease, and much less is known about non-redundant roles in selective cells and tissues. Herein, we set out to comprehensively characterize the cardiac-restricted Heat Shock Protein B-2 (HspB2), which exhibited ischemic cardioprotection in transgenic overexpressing mice including reduced infarct size and maintenance of ATP levels. Global yeast two-hybrid analysis using HspB2 (bait) and a human cardiac library (prey) coupled with co-immunoprecipitation studies for mitochondrial target validation revealed the first HspB2 "cardiac interactome" to contain many myofibril and mitochondrial-binding partners consistent with the overexpression phenotype. This interactome has been submitted to the Biological General Repository for Interaction Datasets (BioGRID). A related sHSP chaperone HspB5 had only partially overlapping binding partners, supporting specificity of the interactome as well as non-redundant roles reported for these sHSPs. Evidence that the cardiac yeast two-hybrid HspB2 interactome targets resident mitochondrial client proteins is consistent with the role of HspB2 in maintaining ATP levels and suggests new chaperone-dependent functions for metabolic homeostasis. One of the HspB2 targets, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), has reported roles in HspB2 associated phenotypes including cardiac ATP production, mitochondrial function, and apoptosis, and was validated as a potential client protein of HspB2 through chaperone assays. From the clientele and phenotypes identified herein, it is tempting to speculate that small molecule activators of HspB2 might be deployed to mitigate mitochondrial related diseases such as cardiomyopathy and neurodegenerative disease.

The Protein Information Resource: an integrated public resource of functional annotation of proteins

PubMed Central

Wu, Cathy H.; Huang, Hongzhan; Arminski, Leslie; Castro-Alvear, Jorge; Chen, Yongxing; Hu, Zhang-Zhi; Ledley, Robert S.; Lewis, Kali C.; Mewes, Hans-Werner; Orcutt, Bruce C.; Suzek, Baris E.; Tsugita, Akira; Vinayaka, C. R.; Yeh, Lai-Su L.; Zhang, Jian; Barker, Winona C.

2002-01-01

The Protein Information Resource (PIR) serves as an integrated public resource of functional annotation of protein data to support genomic/proteomic research and scientific discovery. The PIR, in collaboration with the Munich Information Center for Protein Sequences (MIPS) and the Japan International Protein Information Database (JIPID), produces the PIR-International Protein Sequence Database (PSD), the major annotated protein sequence database in the public domain, containing about 250 000 proteins. To improve protein annotation and the coverage of experimentally validated data, a bibliography submission system is developed for scientists to submit, categorize and retrieve literature information. Comprehensive protein information is available from iProClass, which includes family classification at the superfamily, domain and motif levels, structural and functional features of proteins, as well as cross-references to over 40 biological databases. To provide timely and comprehensive protein data with source attribution, we have introduced a non-redundant reference protein database, PIR-NREF. The database consists of about 800 000 proteins collected from PIR-PSD, SWISS-PROT, TrEMBL, GenPept, RefSeq and PDB, with composite protein names and literature data. To promote database interoperability, we provide XML data distribution and open database schema, and adopt common ontologies. The PIR web site (http://pir.georgetown.edu/) features data mining and sequence analysis tools for information retrieval and functional identification of proteins based on both sequence and annotation information. The PIR databases and other files are also available by FTP (ftp://nbrfa.georgetown.edu/pir_databases). PMID:11752247

Lanthanum Element Induced Imbalance of Mineral Nutrients, HSP 70 Production and DNA-Protein Crosslink, Leading to Hormetic Response of Cell Cycle Progression in Root Tips of Vicia faba L. seedlings.

PubMed

Wang, Chengrun; Shi, Cuie; Liu, Ling; Wang, Chen; Qiao, Wei; Gu, Zhimang; Wang, Xiaorong

2012-01-01

The effects and mechanisms of rare earth elements on plant growth have not been extensively characterized. In the current study, Vicia faba L. seedlings were cultivated in lanthanum (La)-containing solutions for 10 days to investigate the possible effects and mechanisms of La on cell proliferation and root lengthening in roots. The results showed that increasing La levels resulted in abnormal calcium (Ca), Ferrum (Fe) or Potassium (K) contents in the roots. Flow cytometry analysis revealed G1/S and S/G2 arrests in response to La treatments in the root tips. Heat shock protein 70 (HSP 70) production showed a U-shaped dose response to increasing La levels. Consistent with its role in cell cycle regulation, HSP 70 fluctuated in parallel with the S-phase ratios and proliferation index. Furthermore, DNA-protein crosslinks (DPCs) enhanced at higher La concentrations, perhaps involved in blocking cell progression. Taken together, these data provide important insights into the hormetic effects and mechanisms of REE(s) on plant cell proliferation and growth.

Acclimation of Oxygenic Photosynthesis to Iron Starvation Is Controlled by the sRNA IsaR1.

PubMed

Georg, Jens; Kostova, Gergana; Vuorijoki, Linda; Schön, Verena; Kadowaki, Taro; Huokko, Tuomas; Baumgartner, Desirée; Müller, Maximilian; Klähn, Stephan; Allahverdiyeva, Yagut; Hihara, Yukako; Futschik, Matthias E; Aro, Eva-Mari; Hess, Wolfgang R

2017-05-22

Oxygenic photosynthesis crucially depends on proteins that possess Fe 2+ or Fe/S complexes as co-factors or prosthetic groups. Here, we show that the small regulatory RNA (sRNA) IsaR1 (Iron-Stress-Activated RNA 1) plays a pivotal role in acclimation to low-iron conditions. The IsaR1 regulon consists of more than 15 direct targets, including Fe 2+ -containing proteins involved in photosynthetic electron transfer, detoxification of anion radicals, citrate cycle, and tetrapyrrole biogenesis. IsaR1 is essential for maintaining physiological levels of Fe/S cluster biogenesis proteins during iron deprivation. Consequently, IsaR1 affects the acclimation of the photosynthetic apparatus to iron starvation at three levels: (1) directly, via posttranscriptional repression of gene expression; (2) indirectly, via suppression of pigment; and (3) Fe/S cluster biosynthesis. Homologs of IsaR1 are widely conserved throughout the cyanobacterial phylum. We conclude that IsaR1 is a critically important riboregulator. These findings provide a new perspective for understanding the regulation of iron homeostasis in photosynthetic organisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bovine adenovirus 3 core protein precursor pVII localizes to mitochondria, and modulates ATP synthesis, mitochondrial Ca2+ and mitochondrial membrane potential.

PubMed

Anand, Sanjeev K; Gaba, Amit; Singh, Jaswant; Tikoo, Suresh K

2014-02-01

Viruses modulate the functions of mitochondria by translocating viral proteins to the mitochondria. Subcellular fractionation and sensitivity to proteinase K/Triton X-100 treatment of mitochondrial fractions of bovine adenovirus (BAdV)-3-infected/transfected cells suggested that core protein pVII localizes to the mitochondria and contains a functional mitochondrial localization signal. Moreover, mitochondrial localization of BAdV-3 pVII appears to help in the retention of mitochondrial Ca(2+), inducing a significant increase in the levels of ATP and maintaining the mitochondrial membrane potential (MMP) in transfected cells. In contrast, mitochondrial localization of BAdV-3 pVII has no significant effect on the levels of cytoplasmic Ca(2+) and reactive oxygen species production in the transfected cells. Consistent with these results, expression of pVII in transfected cells treated with staurosporine decreased significantly the activation of caspase-3. Our results suggested that BAdV-3 pVII localizes to mitochondria, and interferes with apoptosis by inhibiting loss of the MMP and by increasing mitochondrial Ca(2+) and ATP production.

Substance P activates ADAM9 mRNA expression and induces α-secretase-mediated amyloid precursor protein cleavage.

PubMed

Marolda, R; Ciotti, M T; Matrone, C; Possenti, R; Calissano, P; Cavallaro, S; Severini, C

2012-04-01

Altered levels of Substance P (SP), a neuropeptide endowed with neuroprotective and anti-apoptotic properties, were found in brain areas and spinal fluid of Alzheimer's disease (AD) patients. One of the hallmarks of AD is the abnormal extracellular deposition of neurotoxic beta amyloid (Aβ) peptides, derived from the proteolytic processing of amyloid precursor protein (APP). In the present study, we confirmed, the neurotrophic action of SP in cultured rat cerebellar granule cells (CGCs) and investigated its effects on APP metabolism. Incubation with low (5 mM) potassium induced apoptotic cell death of CGCs and amyloidogenic processing of APP, whereas treatment with SP (200 nM) reverted these effects via NK1 receptors. The non-amyloidogenic effect of SP consisted of reduction of Aβ(1-42), increase of sAPPα and enhanced α-secretase activity, without a significant change in steady-state levels of cellular APP. The intracellular mechanisms whereby SP alters APP metabolism were further investigated by measuring mRNA and/or steady-state protein levels of key enzymes involved with α-, β- and γ-secretase activity. Among them, Adam9, both at the mRNA and protein level, was the only enzyme to be significantly down-regulated following the induction of apoptosis (K5) and up-regulated after SP treatment. In addition to its neuroprotective properties, this study shows that SP is able to stimulate non-amyloidogenic APP processing, thereby reducing the possibility of generation of toxic Aβ peptides in brain. Copyright © 2011 Elsevier Ltd. All rights reserved.

Ameliorating effect of Kalpaamruthaa, a Siddha preparation in adjuvant induced arthritis in rats with reference to changes in proinflammatory cytokines and acute phase proteins.

PubMed

Mythilypriya, Rajendran; Sachdanandam, Palanivelu Shanthi; Sachdanandam, Panchanadam

2009-05-15

As disease initiation and propagation still represents a research question in rheumatoid arthritis (RA), the cytokines play a central role in the inflammatory articular process including the synovial proliferation and cartilage destruction in RA and understanding the role of these cytokines in turn exploits them as therapeutic targets in RA. The present study illustrates the beneficial outcome of the Siddha drug Kalpaamruthaa (KA) in reducing the pathological lesions caused by the proinflammatory cytokines in adjuvant induced arthritis (AIA) in rats. KA consists of Semecarpus anacardium nut milk extract (SA), dried powder of Emblica officinalis fruit and honey. Both SA and KA were administered at dose of 150 mg/kg b.wt. for 14 days after 14 days of adjuvant injection in rats. The protein expressions of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), the levels of acute phase proteins, immunoglobulins and the radiological, histopathological and electron microscopical changes in control and experimental animals were analyzed. Both SA and KA significantly regulated the inflammation in arthritic joints by reducing extracellular matrix degradation and cartilage and bone destruction via down regulating the levels of TNF-alpha and IL-1beta, as well the levels of acute phase proteins with appreciable increase in the levels of immunoglobulins in arthritic rats. Of both the drugs KA exhibited a profound effect than sole treatment of SA and the enhanced effect of KA might be attributed to the combined effect of the flavonoids, tannins, vitamin C and other phytoconstituents present in the drug.

Iron Biofortification and Homeostasis in Transgenic Cassava Roots Expressing the Algal Iron Assimilatory Gene, FEA1

PubMed Central

Ihemere, Uzoma E.; Narayanan, Narayanan N.; Sayre, Richard T.

2012-01-01

We have engineered the tropical root crop cassava (Manihot esculenta) to express the Chlamydomonas reinhardtii iron assimilatory gene, FEA1, in its storage roots with the objective of enhancing the root nutritional qualities. Iron levels in mature cassava storage roots were increased from 10 to 36 ppm in the highest iron accumulating transgenic lines. These iron levels are sufficient to meet the minimum daily requirement for iron in a 500 g meal. Significantly, the expression of the FEA1 gene in storage roots did not alter iron levels in leaves. Transgenic plants also had normal levels of zinc in leaves and roots consistent with the specific uptake of ferrous iron mediated by the FEA1 protein. Relative to wild-type plants, fibrous roots of FEA1 expressing plants had reduced Fe (III) chelate reductase activity consistent with the more efficient uptake of iron in the transgenic plants. We also show that multiple cassava genes involved in iron homeostasis have altered tissue-specific patterns of expression in leaves, stems, and roots of transgenic plants consistent with increased iron sink strength in transgenic roots. These results are discussed in terms of strategies for the iron biofortification of plants. PMID:22993514

Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.

PubMed

Borges, L G; Seifert, R A; Grant, F J; Hart, C E; Disteche, C M; Edelhoff, S; Solca, F F; Lieberman, M A; Lindner, V; Fischer, E H; Lok, S; Bowen-Pope, D F

1996-09-01

We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

Expression of the dopaminergic D1 and D2 receptors in the anterior cingulate cortex in a model of neuropathic pain

PubMed Central

2011-01-01

Background The anterior cingulate cortex (ACC) has been related to the affective component of pain. Dopaminergic mesocortical circuits, including the ACC, are able to inhibit neuropathic nociception measured as autotomy behaviour. We determined the changes in dopamine D1 and D2 (D1R and D2R) receptor expression in the ACC (cg1 and cg2) in an animal model of neuropathic pain. The neuropathic group had noxious heat applied in the right hind paw followed 30 min. later by right sciatic denervation. Autotomy score (AS) was recorded for eight days and subsequently classified in low, medium and high AS groups. The control consisted of naïve animals. A semiquantitative RT-PCR procedure was done to determine mRNA levels for D1R and D2R in cg1 and cg2, and protein levels were measured by Western Blot. Results The results of D1R mRNA in cg1 showed a decrease in all groups. D2R mRNA levels in cg1 decreased in low AS and increased in medium and high AS. Regarding D1R in cg2, there was an increase in all groups. D2R expression levels in cg2 decreased in all groups. In cg1, the D2R mRNA correlated positively with autotomy behaviour. Protein levels of D2R in cg1 increased in all groups but to a higher degree in low AS. In cg2 D2R protein only decreased discretely. D1R protein was not found in either ACC region. Conclusions This is the first evidence of an increase of inhibitory dopaminergic receptor (D2R) mRNA and protein in cg1 in correlation with nociceptive behaviour in a neuropathic model of pain in the rat. PMID:22171983

Identification and validation of quantitative trait loci for seed yield, oil and protein contents in two recombinant inbred line populations of soybean.

PubMed

Wang, Xianzhi; Jiang, Guo-Liang; Green, Marci; Scott, Roy A; Song, Qijian; Hyten, David L; Cregan, Perry B

2014-10-01

Soybean seeds contain high levels of oil and protein, and are the important sources of vegetable oil and plant protein for human consumption and livestock feed. Increased seed yield, oil and protein contents are the main objectives of soybean breeding. The objectives of this study were to identify and validate quantitative trait loci (QTLs) associated with seed yield, oil and protein contents in two recombinant inbred line populations, and to evaluate the consistency of QTLs across different environments, studies and genetic backgrounds. Both the mapping population (SD02-4-59 × A02-381100) and validation population (SD02-911 × SD00-1501) were phenotyped for the three traits in multiple environments. Genetic analysis indicated that oil and protein contents showed high heritabilities while yield exhibited a lower heritability in both populations. Based on a linkage map constructed previously with the mapping population and using composite interval mapping and/or interval mapping analysis, 12 QTLs for seed yield, 16 QTLs for oil content and 11 QTLs for protein content were consistently detected in multiple environments and/or the average data over all environments. Of the QTLs detected in the mapping population, five QTLs for seed yield, eight QTLs for oil content and five QTLs for protein content were confirmed in the validation population by single marker analysis in at least one environment and the average data and by ANOVA over all environments. Eight of these validated QTLs were newly identified. Compared with the other studies, seven QTLs for seed yield, eight QTLs for oil content and nine QTLs for protein content further verified the previously reported QTLs. These QTLs will be useful for breeding higher yield and better quality cultivars, and help effectively and efficiently improve yield potential and nutritional quality in soybean.

Bladder Cancer-associated Protein, a Potential Prognostic Biomarker in Human Bladder Cancer*

PubMed Central

Moreira, José M. A.; Ohlsson, Gita; Gromov, Pavel; Simon, Ronald; Sauter, Guido; Celis, Julio E.; Gromova, Irina

2010-01-01

It is becoming increasingly clear that no single marker will have the sensitivity and specificity necessary to be used on its own for diagnosis/prognosis of tumors. Interpatient and intratumor heterogeneity provides overwhelming odds against the existence of such an ideal marker. With this in mind, our laboratory has been applying a long term systematic approach to identify multiple biomarkers that can be used for clinical purposes. As a result of these studies, we have identified and reported several candidate biomarker proteins that are deregulated in bladder cancer. Following the conceptual biomarker development phases proposed by the Early Detection Research Network, we have taken some of the most promising candidate proteins into postdiscovery validation studies, and here we report on the characterization of one such biomarker, the bladder cancer-associated protein (BLCAP), formerly termed Bc10. To characterize BLCAP protein expression and cellular localization patterns in benign bladder urothelium and urothelial carcinomas (UCs), we used two independent sets of samples from different patient cohorts: a reference set consisting of 120 bladder specimens (formalin-fixed as well as frozen biopsies) and a validation set consisting of 2,108 retrospectively collected UCs with long term clinical follow-up. We could categorize the UCs examined into four groups based on levels of expression and subcellular localization of BLCAP protein and showed that loss of BLCAP expression is associated with tumor progression. The results indicated that increased expression of this protein confers an adverse patient outcome, suggesting that categorization of staining patterns for this protein may have prognostic value. Finally, we applied a combinatorial two-marker discriminator using BLCAP and adipocyte-type fatty acid-binding protein, another UC biomarker previously reported by us, and found that the combination of the two markers correlated more closely with grade and/or stage of disease than the individual markers. The implications of these results in biomarker discovery are discussed. PMID:19783793

Heat‐tolerant Flowering Plants of Active Geothermal Areas in Yellowstone National Park

PubMed Central

STOUT, RICHARD G.; AL‐NIEMI, THAMIR S.

2002-01-01

A broad survey of most of the major geyser basins within Yellowstone National Park (Wyoming, USA) was conducted to identify the flowering plants which tolerate high rhizosphere temperatures (≥40 °C) in geothermally heated environments. Under such conditions, five species of monocots and four species of dicots were repeatedly found. The predominant flowering plants in hot soils (>40 °C at 2–5 cm depth) were grasses, primarily Dichanthelium lanuginosum. Long‐term (weeks to months) rhizosphere temperatures of individual D. lanuginosum above 40 °C were recorded at several different locations, both in the summer and winter. The potential role of heat shock proteins (HSPs) in the apparent adaptation of these plants to chronically high rhizosphere temperatures was examined. Antibodies to cytoplasmic class I small heat shock proteins (sHSPs) and to HSP101 were used in Western immunoblot analyses of protein extracts from plants collected from geothermally heated soils. Relatively high levels of proteins reacting with anti‐sHSP antibodies were consistently detected in root extracts from plants experiencing rhizosphere temperatures above 40 °C, though these proteins were usually not highly expressed in leaf extracts from the same plants. Proteins reacting with antibodies to HSP101 were also present both in leaf and root extracts from plants collected from geothermal soils, but their levels of expression were not as closely related to the degree of heat exposure as those of sHSPs. PMID:12197524

Effect of Inoculum Dosage Aspergillus niger and Rhizopus oryzae mixture with Fermentation Time of Oil Seed Cake (Jatropha curcas L) to the content of Protein and Crude Fiber

NASA Astrophysics Data System (ADS)

Kurniati, T.; Nurlaila, L.; Iim

2017-04-01

Jatropha curcas L already widely cultivated for its seeds pressed oil used as an alternative fuel. This plant productivity per hectare obtained 2.5-5 tonnes of oil/ha / year and jatropha seed cake from 5.5 to 9.5 tonnes/ha/year, nutrient content of Jatropha curcas seed L potential to be used as feed material, However, the constraints faced was the low crude protein and high crude protein. The purpose of the research was to determine the dosage of inoculum and fermentation time of Jatropha seed cake by a mixture of Aspergillus niger and Rhizopus oryzae on crude protein and crude fibre. The study was conducted by an experimental method using a Completely Randomised Design (CRD) factorial design (3×3). The treatment consisted of a mixture of three dosage levels of Aspergillus niger and Rhizopus oryzae (= 0.2% d1, d2 and d3 = 0.3% = 0.4%) and three levels of fermentation time (w1 = 72 hours, 96 hours and w2 = w3 = 120 hours) each repeated three times. The parameters measured were crude protein and crude fibre. The results showed that dosages of 0.3% (Aspergillus niger Rhizopus oryzae 0.15% and 0.15%) and 72 hours (d2w1) is the dosage and the optimal time to generate the highest crude protein content of 21.11% and crude fibre amounted to 21.36%.

Reproducible Tissue Homogenization and Protein Extraction for Quantitative Proteomics Using MicroPestle-Assisted Pressure-Cycling Technology.

PubMed

Shao, Shiying; Guo, Tiannan; Gross, Vera; Lazarev, Alexander; Koh, Ching Chiek; Gillessen, Silke; Joerger, Markus; Jochum, Wolfram; Aebersold, Ruedi

2016-06-03

The reproducible and efficient extraction of proteins from biopsy samples for quantitative analysis is a critical step in biomarker and translational research. Recently, we described a method consisting of pressure-cycling technology (PCT) and sequential windowed acquisition of all theoretical fragment ions-mass spectrometry (SWATH-MS) for the rapid quantification of thousands of proteins from biopsy-size tissue samples. As an improvement of the method, we have incorporated the PCT-MicroPestle into the PCT-SWATH workflow. The PCT-MicroPestle is a novel, miniaturized, disposable mechanical tissue homogenizer that fits directly into the microTube sample container. We optimized the pressure-cycling conditions for tissue lysis with the PCT-MicroPestle and benchmarked the performance of the system against the conventional PCT-MicroCap method using mouse liver, heart, brain, and human kidney tissues as test samples. The data indicate that the digestion of the PCT-MicroPestle-extracted proteins yielded 20-40% more MS-ready peptide mass from all tissues tested with a comparable reproducibility when compared to the conventional PCT method. Subsequent SWATH-MS analysis identified a higher number of biologically informative proteins from a given sample. In conclusion, we have developed a new device that can be seamlessly integrated into the PCT-SWATH workflow, leading to increased sample throughput and improved reproducibility at both the protein extraction and proteomic analysis levels when applied to the quantitative proteomic analysis of biopsy-level samples.

Adenovirus Death Protein (ADP) Is Required for Lytic Infection of Human Lymphocytes

PubMed Central

Murali, V. K.; Ornelles, D. A.; Gooding, L. R.; Wilms, H. T.; Huang, W.; Tollefson, A. E.; Wold, W. S. M.

2014-01-01

The adenovirus death protein (ADP) is expressed at late times during a lytic infection of species C adenoviruses. ADP promotes the release of progeny virus by accelerating the lysis and death of the host cell. Since some human lymphocytes survive while maintaining a persistent infection with species C adenovirus, we compared ADP expression in these cells with ADP expression in lymphocytes that proceed with a lytic infection. Levels of ADP were low in KE37 and BJAB cells, which support a persistent infection. In contrast, levels of ADP mRNA and protein were higher in Jurkat cells, which proceed with a lytic infection. Epithelial cells infected with an ADP-overexpressing virus died more quickly than epithelial cells infected with an ADP-deleted virus. However, KE37, and BJAB cells remained viable after infection with the ADP-overexpressing virus. Although the levels of ADP mRNA increased in KE37 and BJAB cells infected with the ADP-overexpressing virus, the fraction of cells with detectable ADP was unchanged, suggesting that the control of ADP expression differs between epithelial and lymphocytic cells. When infected with an ADP-deleted adenovirus, Jurkat cells survived and maintained viral DNA for greater than 1 month. These findings are consistent with the notion that the level of ADP expression determines whether lymphocytic cells proceed with a lytic or a persistent adenovirus infection. PMID:24198418

Inclusion of Cocoa as a Dietary Supplement Represses Expression of Inflammatory Proteins in Spinal Trigeminal Nucleus in Response to Chronic Trigeminal Nerve Stimulation

PubMed Central

Cady, Ryan J.; Denson, Jennifer E.; Durham, Paul L.

2013-01-01

Scope Central sensitization is implicated in the pathology of temporomandibular joint disorder (TMD) and other types of orofacial pain. We investigated the effects of dietary cocoa on expression of proteins involved in the development of central sensitization in the spinal trigeminal nucleus (STN) in response to inflammatory stimulation of trigeminal nerves. Methods and results Male Sprague Dawley rats were fed either a control diet or an isocaloric diet consisting of 10% cocoa powder 14 days prior to bilateral injection of complete Freund’s adjuvant (CFA) into the temporomandibular joint to promote prolonged activation of trigeminal ganglion neurons and glia. While dietary cocoa stimulated basal expression of GLAST and MKP-1 when compared to animals on a normal diet, cocoa suppressed basal calcitonin gene-related peptide levels in the STN. CFA-stimulated levels of protein kinase A, P2X3, P-p38, GFAP, and OX-42, whose elevated levels in the STN are implicated in central sensitization, were repressed to near control levels in animals on a cocoa enriched diet. Similarly, dietary cocoa repressed CFA-stimulated inflammatory cytokine expression. Conclusion Based on our findings, we speculate that cocoa enriched diets could be beneficial as a natural therapeutic option for TMD and other chronic orofacial pain conditions. PMID:23576361

Impaired thymic selection in mice expressing altered levels of the SLP-76 adaptor protein.

PubMed

Ramsey, Kimberley; Luckashenak, Nancy; Koretzky, Gary A; Clements, James L

2008-02-01

Intracellular signaling initiated by ligation of the TCR influences cell fate at multiple points during the lifespan of a T cell. This is especially evident during thymic selection, where the nature of TCR-dependent signaling helps to establish a MHC-restricted, self-tolerant T cell repertoire. The Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) adaptor protein is a required intermediate in multiple signaling pathways triggered by TCR engagement, several of which have been implicated in dictating the outcome of thymic selection (e.g., intracellular calcium flux and activation of ERK family MAPKs). To determine if thymocyte maturation and selection at later stages of development are sensitive to perturbations in SLP-76 levels, we analyzed these crucial events using several transgenic (Tg) lines of mice expressing altered levels of SLP-76 in the thymus. In Tg mice expressing low levels of SLP-76 in preselection thymocytes, the CD4:CD8 ratio in the thymus and spleen was skewed in a manner consistent with impaired selection and/or maturation of CD4+ thymocytes. Low SLP-76 expression also correlated with reduced CD5 expression on immature thymocytes, consistent with reduced TCR signaling potential. In contrast, reconstitution of SLP-76 at higher levels resulted in normal thymic CD5 expression and CD4:CD8 ratios in the thymus and periphery. It is curious that thymic deletion of TCR-Tg (HY) thymocytes was markedly impaired in both lines of Tg-reconstituted SLP-76-/- mice. Studies using chimeric mice indicate that the defect in deletion of HY+ thymocytes is intrinsic to the developing thymocyte, suggesting that maintenance of sufficient SLP-76 expression from the endogenous locus is a key element in the selection process.

Th1 cytokine-induced syndecan-4 shedding by airway smooth muscle cells is dependent on mitogen-activated protein kinases.

PubMed

Tan, Xiahui; Khalil, Najwa; Tesarik, Candice; Vanapalli, Karunasri; Yaputra, Viki; Alkhouri, Hatem; Oliver, Brian G G; Armour, Carol L; Hughes, J Margaret

2012-04-01

In asthma, airway smooth muscle (ASM) chemokine secretion can induce mast cell recruitment into the airways. The functions of the mast cell chemoattractant CXCL10, and other chemokines, are regulated by binding to heparan sulphates such as syndecan-4. This study is the first demonstration that airway smooth muscle cells (ASMC) from people with and without asthma express and shed syndecan-4 under basal conditions. Syndecan-4 shedding was enhanced by stimulation for 24 h with the Th1 cytokines interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α), but not interferon-γ (IFNγ), nor the Th2 cytokines IL-4 and IL-13. ASMC stimulation with IL-1β, TNF-α, and IFNγ (cytomix) induced the highest level of syndecan-4 shedding. Nonasthmatic and asthmatic ASM cell-associated syndecan-4 protein expression was also increased by TNF-α or cytomix at 4-8 h, with the highest levels detected in cytomix-stimulated asthmatic cells. Cell-associated syndecan-4 levels were decreased by 24 h, whereas shedding remained elevated at 24 h, consistent with newly synthesized syndecan-4 being shed. Inhibition of ASMC matrix metalloproteinase-2 did not prevent syndecan-4 shedding, whereas inhibition of ERK MAPK activation reduced shedding from cytomix-stimulated ASMC. Although ERK inhibition had no effect on syndecan-4 mRNA levels stimulated by cytomix, it did cause an increase in cell-associated syndecan-4 levels, consistent with the shedding being inhibited. In conclusion, ASMC produce and shed syndecan-4 and although this is increased by the Th1 cytokines, the MAPK ERK only regulates shedding. ASMC syndecan-4 production during Th1 inflammatory conditions may regulate chemokine activity and mast cell recruitment to the ASM in asthma.

Analysis of the Aspergillus fumigatus proteome reveals metabolic changes and the activation of the pseurotin A biosynthesis gene cluster in response to hypoxia.

PubMed

Vödisch, Martin; Scherlach, Kirstin; Winkler, Robert; Hertweck, Christian; Braun, Hans-Peter; Roth, Martin; Haas, Hubertus; Werner, Ernst R; Brakhage, Axel A; Kniemeyer, Olaf

2011-05-06

The mold Aspergillus fumigatus is the most important airborne fungal pathogen. Adaptation to hypoxia represents an important virulence attribute for A. fumigatus. Therefore, we aimed at obtaining a comprehensive overview about this process on the proteome level. To ensure highly reproducible growth conditions, an oxygen-controlled, glucose-limited chemostat cultivation was established. Two-dimensional gel electrophoresis analysis of mycelial and mitochondrial proteins as well as two-dimensional Blue Native/SDS-gel separation of mitochondrial membrane proteins led to the identification of 117 proteins with an altered abundance under hypoxic in comparison to normoxic conditions. Hypoxia induced an increased activity of glycolysis, the TCA-cycle, respiration, and amino acid metabolism. Consistently, the cellular contents in heme, iron, copper, and zinc increased. Furthermore, hypoxia induced biosynthesis of the secondary metabolite pseurotin A as demonstrated at proteomic, transcriptional, and metabolite levels. The observed and so far not reported stimulation of the biosynthesis of a secondary metabolite by oxygen depletion may also affect the survival of A. fumigatus in hypoxic niches of the human host. Among the proteins so far not implicated in hypoxia adaptation, an NO-detoxifying flavohemoprotein was one of the most highly up-regulated proteins which indicates a link between hypoxia and the generation of nitrosative stress in A. fumigatus.

A proteomic style approach to characterize a grass mix product reveals potential immunotherapeutic benefit.

PubMed

Bullimore, Alan; Swan, Nicola; Alawode, Wemimo; Skinner, Murray

2011-09-01

Grass allergy immunotherapies often consist of a mix of different grass extracts, each containing several proteins of different physiochemical properties; however, the subtle contributions of each protein are difficult to elucidate. This study aimed to identify and characterize the group 1 and 5 allergens in a 13 grass extract and to standardize the extraction method. The grass pollens were extracted in isolation and pooled and also in combination and analyzed using a variety of techniques including enzyme-linked immunosorbent assay, liquid chromatog-raphy-mass spectrometry, and sodium dodecyl sulfate-polyacrylam-ide gel electrophoresis. Gold-staining and IgE immunoblotting revealed a high degree of homology of protein bands between the 13 species and the presence of a densely stained doublet at 25-35 kD along with protein bands at approximately 12.5, 17, and 50 kD. The doublet from each grass species demonstrated a high level of group 1 and 5 interspecies homology. However, there were a number of bands unique to specific grasses consistent with evolutionary change and indicative that a grass mix immunotherapeutic could be considered broad spectrum. Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis and IgE immunoblotting showed all 13 grasses share a high degree of homology, particularly in terms of group 1 and 5 allergens. IgE and IgG enzyme-linked immunosorbent assay potencies were shown to be independent of extraction method.

BMI was found to be a consistent determinant related to misreporting of energy, protein and potassium intake using self-report and duplicate portion methods.

PubMed

Trijsburg, Laura; Geelen, Anouk; Hollman, Peter Ch; Hulshof, Paul Jm; Feskens, Edith Jm; Van't Veer, Pieter; Boshuizen, Hendriek C; de Vries, Jeanne Hm

2017-03-01

As misreporting, mostly under-reporting, of dietary intake is a generally known problem in nutritional research, we aimed to analyse the association between selected determinants and the extent of misreporting by the duplicate portion method (DP), 24 h recall (24hR) and FFQ by linear regression analysis using the biomarker values as unbiased estimates. For each individual, two DP, two 24hR, two FFQ and two 24 h urinary biomarkers were collected within 1·5 years. Also, for sixty-nine individuals one or two doubly labelled water measurements were obtained. The associations of basic determinants (BMI, gender, age and level of education) with misreporting of energy, protein and K intake of the DP, 24hR and FFQ were evaluated using linear regression analysis. Additionally, associations between other determinants, such as physical activity and smoking habits, and misreporting were investigated. The Netherlands. One hundred and ninety-seven individuals aged 20-70 years. Higher BMI was associated with under-reporting of dietary intake assessed by the different dietary assessment methods for energy, protein and K, except for K by DP. Men tended to under-report protein by the DP, FFQ and 24hR, and persons of older age under-reported K but only by the 24hR and FFQ. When adjusted for the basic determinants, the other determinants did not show a consistent association with misreporting of energy or nutrients and by the different dietary assessment methods. As BMI was the only consistent determinant of misreporting, we conclude that BMI should always be taken into account when assessing and correcting dietary intake.

Reduced protein oxidation in Wistar rats supplemented with marine ω3 PUFAs.

PubMed

Méndez, Lucía; Pazos, Manuel; Gallardo, José M; Torres, Josep L; Pérez-Jiménez, Jara; Nogués, Rosa; Romeu, Marta; Medina, Isabel

2013-02-01

The potential effects of various dietary eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6) ratios (1:1, 2:1, and 1:2, respectively) on protein redox states from plasma, kidney, skeletal muscle, and liver were investigated in Wistar rats. Dietary fish oil groups were compared with animals fed soybean and linseed oils, vegetable oils enriched in ω6 linoleic acid (LA; 18:2) and ω3 α-linolenic acid (ALA; 18:3), respectively. Fish oil treatments were effective at reducing the level of total fatty acids in plasma and enriching the plasmatic free fatty acid fraction and erythrocyte membranes in EPA and DHA. A proteomic approach consisting of fluorescein 5-thiosemicarbazide (FTSC) labeling of protein carbonyls, FTSC intensity visualization on 1-DE or 2-DE gels, and protein identification by MS/MS was used for the protein oxidation assessment. Albumin was found to be the most carbonylated protein in plasma for all dietary groups, and its oxidation level was significantly modulated by dietary interventions. Supplementation with an equal EPA:DHA ratio (1:1) showed the lowest oxidation score for plasma albumin, followed in increasing order of carbonylation by 1:2 <2:1 ≈ linseed < soybean. Oxidation patterns of myofibrillar skeletal muscle proteins and cytosolic proteins from kidney and liver also indicated a protective effect on proteins for the fish oil treatments, the 1:1 ratio exhibiting the lowest protein oxidation scores. The effect of fish oil treatments at reducing carbonylation on specific proteins from plasma (albumin), skeletal muscle (actin), and liver (albumin, argininosuccinate synthetase, 3-α-hydroxysteroid dehydrogenase) was remarkable. This investigation highlights the efficiency of dietary fish oil at reducing in vivo oxidative damage of proteins compared to oils enriched in the 18-carbon polyunsaturated fatty acids ω3 ALA and ω6 LA, and such antioxidant activity may differ among different fish oil sources because of variations in EPA/DHA content. Copyright © 2012 Elsevier Inc. All rights reserved.

Drosophila Fatty Acid Transport Protein Regulates Rhodopsin-1 Metabolism and Is Required for Photoreceptor Neuron Survival

PubMed Central

Dourlen, Pierre; Bertin, Benjamin; Chatelain, Gilles; Robin, Marion; Napoletano, Francesco; Roux, Michel J.; Mollereau, Bertrand

2012-01-01

Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance. PMID:22844251

Trichomonas vaginalis vast BspA-like gene family: evidence for functional diversity from structural organisation and transcriptomics

PubMed Central

2010-01-01

Background Trichomonas vaginalis is the most common non-viral human sexually transmitted pathogen and importantly, contributes to facilitating the spread of HIV. Yet very little is known about its surface and secreted proteins mediating interactions with, and permitting the invasion and colonisation of, the host mucosa. Initial annotations of T. vaginalis genome identified a plethora of candidate extracellular proteins. Results Data mining of the T. vaginalis genome identified 911 BspA-like entries (TvBspA) sharing TpLRR-like leucine-rich repeats, which represent the largest gene family encoding potential extracellular proteins for the pathogen. A broad range of microorganisms encoding BspA-like proteins was identified and these are mainly known to live on mucosal surfaces, among these T. vaginalis is endowed with the largest gene family. Over 190 TvBspA proteins with inferred transmembrane domains were characterised by a considerable structural diversity between their TpLRR and other types of repetitive sequences and two subfamilies possessed distinct classic sorting signal motifs for endocytosis. One TvBspA subfamily also shared a glycine-rich protein domain with proteins from Clostridium difficile pathogenic strains and C. difficile phages. Consistent with the hypothesis that TvBspA protein structural diversity implies diverse roles, we demonstrated for several TvBspA genes differential expression at the transcript level in different growth conditions. Identified variants of repetitive segments between several TvBspA paralogues and orthologues from two clinical isolates were also consistent with TpLRR and other repetitive sequences to be functionally important. For one TvBspA protein cell surface expression and antibody responses by both female and male T. vaginalis infected patients were also demonstrated. Conclusions The biased mucosal habitat for microbial species encoding BspA-like proteins, the characterisation of a vast structural diversity for the TvBspA proteins, differential expression of a subset of TvBspA genes and the cellular localisation and immunological data for one TvBspA; all point to the importance of the TvBspA proteins to various aspects of T. vaginalis pathobiology at the host-pathogen interface. PMID:20144183

Molecular Dynamics Simulations, Challenges and Opportunities: A Biologist's Prospective.

PubMed

Kumari, Indu; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

2017-08-30

Molecular dynamics (MD) is a computational technique which is used to study biomolecules in virtual environment. Each of the constituent atoms represents a particle and hence the biomolecule embodies a multi-particle mechanical system analyzed within a simulation box during MD analysis. The potential energies of the atoms are explained by a mathematical expression consisting of different forces and space parameters. There are various software and force fields that have been developed for MD studies of the biomolecules. MD analysis has unravelled the various biological mechanisms (protein folding/unfolding, protein-small molecule interactions, protein-protein interactions, DNA/RNA-protein interactions, proteins embedded in membrane, lipid-lipid interactions, drug transport etc.) operating at the atomic and molecular levels. However, there are still some parameters including torsions in amino acids, carbohydrates (whose structure is extended and not well defined like that of proteins) and single stranded nucleic acids for which the force fields need further improvement, although there are several workers putting in constant efforts in these directions. The existing force fields are not efficient for studying the crowded environment inside the cells, since these interactions involve multiple factors in real time. Therefore, the improved force fields may provide the opportunities for their wider applications on the complex biosystems in diverse cellular conditions. In conclusion, the intervention of MD in the basic sciences involving interdisciplinary approaches will be helpful for understanding many fundamental biological and physiological processes at the molecular levels that may be further applied in various fields including biotechnology, fisheries, sustainable agriculture and biomedical research. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Sexual Conflict and Seminal Fluid Proteins: A Dynamic Landscape of Sexual Interactions

PubMed Central

Sirot, Laura K.; Wong, Alex; Chapman, Tracey; Wolfner, Mariana F.

2015-01-01

Sexual reproduction requires coordinated contributions from both sexes to proceed efficiently. However, the reproductive strategies that the sexes adopt often have the potential to give rise to sexual conflict because they can result in divergent, sex-specific costs and benefits. These conflicts can occur at many levels, from molecular to behavioral. Here, we consider sexual conflict mediated through the actions of seminal fluid proteins. These proteins provide many excellent examples in which to trace the operation of sexual conflict from molecules through to behavior. Seminal fluid proteins are made by males and provided to females during mating. As agents that can modulate egg production at several steps, as well as reproductive behavior, sperm “management,” and female feeding, activity, and longevity, the actions of seminal proteins are prime targets for sexual conflict. We review these actions in the context of sexual conflict. We discuss genomic signatures in seminal protein (and related) genes that are consistent with current or previous sexual conflict. Finally, we note promising areas for future study and highlight real-world practical situations that will benefit from understanding the nature of sexual conflicts mediated by seminal proteins. PMID:25502515

Sexual conflict and seminal fluid proteins: a dynamic landscape of sexual interactions.

PubMed

Sirot, Laura K; Wong, Alex; Chapman, Tracey; Wolfner, Mariana F

2014-12-11

Sexual reproduction requires coordinated contributions from both sexes to proceed efficiently. However, the reproductive strategies that the sexes adopt often have the potential to give rise to sexual conflict because they can result in divergent, sex-specific costs and benefits. These conflicts can occur at many levels, from molecular to behavioral. Here, we consider sexual conflict mediated through the actions of seminal fluid proteins. These proteins provide many excellent examples in which to trace the operation of sexual conflict from molecules through to behavior. Seminal fluid proteins are made by males and provided to females during mating. As agents that can modulate egg production at several steps, as well as reproductive behavior, sperm "management," and female feeding, activity, and longevity, the actions of seminal proteins are prime targets for sexual conflict. We review these actions in the context of sexual conflict. We discuss genomic signatures in seminal protein (and related) genes that are consistent with current or previous sexual conflict. Finally, we note promising areas for future study and highlight real-world practical situations that will benefit from understanding the nature of sexual conflicts mediated by seminal proteins. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

Cloning, characterization, expression and comparative analysis of pig Golgi membrane sphingomyelin synthase 1.

PubMed

Guillén, Natalia; Navarro, María A; Surra, Joaquín C; Arnal, Carmen; Fernández-Juan, Marta; Cebrián-Pérez, Jose Alvaro; Osada, Jesús

2007-02-15

Pig sphingomyelin synthase 1 (SMS1) cDNA was cloned, characterized and compared to the human ortholog. Porcine protein consists of 413 amino acids and displays a 97% sequence identity with human protein. A phylogenic tree of proteins reveals that porcine SMS1 is more closely related to bovine and rodent proteins than to human. Analysis of protein mass was higher than the theoretical prediction based on amino acid sequence suggesting a kind of posttranslational modification. Quantitative representation of tissue distribution obtained by real-time RT-PCR showed that it was widely expressed although important variations in levels were obtained among organs. Thus, the cardiovascular system, especially the heart, showed the highest value of all the tissues studied. Regional differences of expression were observed in the central nervous system and intestinal tract. Analysis of the hepatic mRNA and protein expressions of SMS1 following turpentine treatment revealed a progressive decrease in the former paralleled by a decrease in the protein concentration. These findings indicate the variation in expression in the different tissues might suggest a different requirement of Golgi sphingomyelin for the specific function in each organ and a regulation of the enzyme in response to turpentine-induced hepatic injury.

Atomic-level description of ubiquitin folding

PubMed Central

Piana, Stefano; Lindorff-Larsen, Kresten; Shaw, David E.

2013-01-01

Equilibrium molecular dynamics simulations, in which proteins spontaneously and repeatedly fold and unfold, have recently been used to help elucidate the mechanistic principles that underlie the folding of fast-folding proteins. The extent to which the conclusions drawn from the analysis of such proteins, which fold on the microsecond timescale, apply to the millisecond or slower folding of naturally occurring proteins is, however, unclear. As a first attempt to address this outstanding issue, we examine here the folding of ubiquitin, a 76-residue-long protein found in all eukaryotes that is known experimentally to fold on a millisecond timescale. Ubiquitin folding has been the subject of many experimental studies, but its slow folding rate has made it difficult to observe and characterize the folding process through all-atom molecular dynamics simulations. Here we determine the mechanism, thermodynamics, and kinetics of ubiquitin folding through equilibrium atomistic simulations. The picture emerging from the simulations is in agreement with a view of ubiquitin folding suggested from previous experiments. Our findings related to the folding of ubiquitin are also consistent, for the most part, with the folding principles derived from the simulation of fast-folding proteins, suggesting that these principles may be applicable to a wider range of proteins. PMID:23503848

Circadian clock-dependent and -independent rhythmic proteomes implement distinct diurnal functions in mouse liver.

PubMed

Mauvoisin, Daniel; Wang, Jingkui; Jouffe, Céline; Martin, Eva; Atger, Florian; Waridel, Patrice; Quadroni, Manfredo; Gachon, Frédéric; Naef, Felix

2014-01-07

Diurnal oscillations of gene expression controlled by the circadian clock underlie rhythmic physiology across most living organisms. Although such rhythms have been extensively studied at the level of transcription and mRNA accumulation, little is known about the accumulation patterns of proteins. Here, we quantified temporal profiles in the murine hepatic proteome under physiological light-dark conditions using stable isotope labeling by amino acids quantitative MS. Our analysis identified over 5,000 proteins, of which several hundred showed robust diurnal oscillations with peak phases enriched in the morning and during the night and related to core hepatic physiological functions. Combined mathematical modeling of temporal protein and mRNA profiles indicated that proteins accumulate with reduced amplitudes and significant delays, consistent with protein half-life data. Moreover, a group comprising about one-half of the rhythmic proteins showed no corresponding rhythmic mRNAs, indicating significant translational or posttranslational diurnal control. Such rhythms were highly enriched in secreted proteins accumulating tightly during the night. Also, these rhythms persisted in clock-deficient animals subjected to rhythmic feeding, suggesting that food-related entrainment signals influence rhythms in circulating plasma factors.

Circadian clock-dependent and -independent rhythmic proteomes implement distinct diurnal functions in mouse liver

PubMed Central

Mauvoisin, Daniel; Wang, Jingkui; Jouffe, Céline; Martin, Eva; Atger, Florian; Waridel, Patrice; Quadroni, Manfredo; Gachon, Frédéric; Naef, Felix

2014-01-01

Diurnal oscillations of gene expression controlled by the circadian clock underlie rhythmic physiology across most living organisms. Although such rhythms have been extensively studied at the level of transcription and mRNA accumulation, little is known about the accumulation patterns of proteins. Here, we quantified temporal profiles in the murine hepatic proteome under physiological light–dark conditions using stable isotope labeling by amino acids quantitative MS. Our analysis identified over 5,000 proteins, of which several hundred showed robust diurnal oscillations with peak phases enriched in the morning and during the night and related to core hepatic physiological functions. Combined mathematical modeling of temporal protein and mRNA profiles indicated that proteins accumulate with reduced amplitudes and significant delays, consistent with protein half-life data. Moreover, a group comprising about one-half of the rhythmic proteins showed no corresponding rhythmic mRNAs, indicating significant translational or posttranslational diurnal control. Such rhythms were highly enriched in secreted proteins accumulating tightly during the night. Also, these rhythms persisted in clock-deficient animals subjected to rhythmic feeding, suggesting that food-related entrainment signals influence rhythms in circulating plasma factors. PMID:24344304

Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease

PubMed Central

Bender, Tom; Lewrenz, Ilka; Franken, Sebastian; Baitzel, Catherina; Voos, Wolfgang

2011-01-01

Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria. PMID:21209324

APC+/− alters colonic fibroblast proteome in FAP

PubMed Central

Dixon, Maketa P.; Blagoi, Elena L.; Nicolas, Emmanuelle; Seeholzer, Steven H.; Cheng, David; He, Yin A.; Coudry, Renata A.; Howard, Sharon D.; Riddle, Dawn M.; Cooper, Harry S.; Boman, Bruce M.; Conrad, Peggy; Crowell, James A.; Bellacosa, Alfonso; Knudson, Alfred; Yeung, Anthony T.; Kopelovich, Levy

2011-01-01

Here we compared the proteomes of primary fibroblast cultures derived from morphologically normal colonic mucosa of familial adenomatous polyposis (FAP) patients with those obtained from unaffected controls. The expression signature of about 19% of total fibroblast proteins separates FAP mutation carriers from unaffected controls (P < 0.01). More than 4,000 protein spots were quantified by 2D PAGE analysis, identifying 368 non-redundant proteins and 400 of their isoforms. Specifically, all three classes of cytoskeletal filaments and their regulatory proteins were altered as were oxidative stress response proteins. Given that FAP fibroblasts showed heightened sensitivity to transformation by KiMSV and SV40 including elevated levels of the p53 protein, events controlled in large measure by the Ras suppressor protein-1 (RSU-1) and oncogenic DJ-1, here we show decreased RSU1 and augmented DJ-1 expression in both fibroblasts and crypt-derived epithelial cells from morphologically normal colonic mucosa of FAP gene-carriers. The results indicate that heterozygosity for a mutant APC tumor suppressor gene alters the proteomes of both colon-derived normal fibroblasts in a gene-specific manner, consistent with a “one-hit” effect. PMID:21411865

There is Diversity in Disorder-"In all Chaos there is a Cosmos, in all Disorder a Secret Order".

PubMed

Nielsen, Jakob T; Mulder, Frans A A

2016-01-01

The protein universe consists of a continuum of structures ranging from full order to complete disorder. As the structured part of the proteome has been intensively studied, stably folded proteins are increasingly well documented and understood. However, proteins that are fully, or in large part, disordered are much less well characterized. Here we collected NMR chemical shifts in a small database for 117 protein sequences that are known to contain disorder. We demonstrate that NMR chemical shift data can be brought to bear as an exquisite judge of protein disorder at the residue level, and help in validation. With the help of secondary chemical shift analysis we demonstrate that the proteins in the database span the full spectrum of disorder, but still, largely segregate into two classes; disordered with small segments of order scattered along the sequence, and structured with small segments of disorder inserted between the different structured regions. A detailed analysis reveals that the distribution of order/disorder along the sequence shows a complex and asymmetric distribution, that is highly protein-dependent. Access to ratified training data further suggests an avenue to improving prediction of disorder from sequence.

Identifying Key Attributes for Protein Beverages.

PubMed

Oltman, A E; Lopetcharat, K; Bastian, E; Drake, M A

2015-06-01

This study identified key attributes of protein beverages and evaluated effects of priming on liking of protein beverages. An adaptive choice-based conjoint study was conducted along with Kano analysis to gain insight on protein beverage consumers (n = 432). Attributes evaluated included label claim, protein type, amount of protein, carbohydrates, sweeteners, and metabolic benefits. Utility scores for levels and importance scores for attributes were determined. Subsequently, two pairs of clear acidic whey protein beverages were manufactured that differed by age of protein source or the amount of whey protein per serving. Beverages were evaluated by 151 consumers on two occasions with or without priming statements. One priming statement declared "great flavor," the other priming statement declared 20 g protein per serving. A two way analysis of variance was applied to discern the role of each priming statement. The most important attribute for protein beverages was sweetener type, followed by amount of protein, followed by type of protein followed by label claim. Beverages with whey protein, naturally sweetened, reduced sugar and ≥15 g protein per serving were most desired. Three consumer clusters were identified, differentiated by their preferences for protein type, sweetener and amount of protein. Priming statements positively impacted concept liking (P < 0.05) but had no effect on overall liking (P > 0.05). Consistent with trained panel profiles of increased cardboard flavor with higher protein content, consumers liked beverages with 10 g protein more than beverages with 20 g protein (6.8 compared with 5.7, P < 0.05). Protein beverages must have desirable flavor for wide consumer appeal. © 2015 Institute of Food Technologists®

A thermodynamically consistent model of the post-translational Kai circadian clock

PubMed Central

Lubensky, David K.; ten Wolde, Pieter Rein

2017-01-01

The principal pacemaker of the circadian clock of the cyanobacterium S. elongatus is a protein phosphorylation cycle consisting of three proteins, KaiA, KaiB and KaiC. KaiC forms a homohexamer, with each monomer consisting of two domains, CI and CII. Both domains can bind and hydrolyze ATP, but only the CII domain can be phosphorylated, at two residues, in a well-defined sequence. While this system has been studied extensively, how the clock is driven thermodynamically has remained elusive. Inspired by recent experimental observations and building on ideas from previous mathematical models, we present a new, thermodynamically consistent, statistical-mechanical model of the clock. At its heart are two main ideas: i) ATP hydrolysis in the CI domain provides the thermodynamic driving force for the clock, switching KaiC between an active conformational state in which its phosphorylation level tends to rise and an inactive one in which it tends to fall; ii) phosphorylation of the CII domain provides the timer for the hydrolysis in the CI domain. The model also naturally explains how KaiA, by acting as a nucleotide exchange factor, can stimulate phosphorylation of KaiC, and how the differential affinity of KaiA for the different KaiC phosphoforms generates the characteristic temporal order of KaiC phosphorylation. As the phosphorylation level in the CII domain rises, the release of ADP from CI slows down, making the inactive conformational state of KaiC more stable. In the inactive state, KaiC binds KaiB, which not only stabilizes this state further, but also leads to the sequestration of KaiA, and hence to KaiC dephosphorylation. Using a dedicated kinetic Monte Carlo algorithm, which makes it possible to efficiently simulate this system consisting of more than a billion reactions, we show that the model can describe a wealth of experimental data. PMID:28296888

LC-QTOF-MS identification of porcine-specific peptide in heat treated pork identifies candidate markers for meat species determination.

PubMed

Sarah, S A; Faradalila, W N; Salwani, M S; Amin, I; Karsani, S A; Sazili, A Q

2016-05-15

The purpose of this study was to identify porcine-specific peptide markers from thermally processed meat that could differentiate pork from beef, chevon and chicken meat. In the initial stage, markers from tryptic digested protein of chilled, boiled and autoclaved pork were identified using LC-QTOF-MS. An MRM method was then established for verification. A thorough investigation of LC-QTOF-MS data showed that only seven porcine-specific peptides were consistently detected. Among these peptides, two were derived from lactate dehydrogenase, one from creatine kinase, and four from serum albumin protein. However, MRM could only detect four peptides (EVTEFAK, LVVITAGAR, FVIER and TVLGNFAAFVQK) that were consistently present in pork samples. In conclusion, meat species determination through a tandem mass spectrometry platform shows high potential in providing scientifically valid and reliable results even at peptide level. Besides, the specificity and selectivity offered by the proteomics approach also provide a robust platform for Halal authentication. Copyright © 2015 Elsevier Ltd. All rights reserved.

Immunotherapy Added to Antibiotic Treatment Reduces Relapse of Disease in a Mouse Model of Tuberculosis.

PubMed

Mourik, Bas C; Leenen, Pieter J M; de Knegt, Gerjo J; Huizinga, Ruth; van der Eerden, Bram C J; Wang, Jinshan; Krois, Charles R; Napoli, Joseph L; Bakker-Woudenberg, Irma A J M; de Steenwinkel, Jurriaan E M

2017-02-01

Immune-modulating drugs that target myeloid-derived suppressor cells or stimulate natural killer T cells have been shown to reduce mycobacterial loads in tuberculosis (TB). We aimed to determine if a combination of these drugs as adjunct immunotherapy to conventional antibiotic treatment could also increase therapeutic efficacy against TB. In our model of pulmonary TB in mice, we applied treatment with isoniazid, rifampicin, and pyrazinamide for 13 weeks alone or combined with immunotherapy consisting of all-trans retinoic acid, 1,25(OH) 2 -vitamin D3, and α-galactosylceramide. Outcome parameters were mycobacterial load during treatment (therapeutic activity) and 13 weeks after termination of treatment (therapeutic efficacy). Moreover, cellular changes were analyzed using flow cytometry and cytokine expression was assessed at the mRNA and protein levels. Addition of immunotherapy was associated with lower mycobacterial loads after 5 weeks of treatment and significantly reduced relapse of disease after a shortened 13-week treatment course compared with antibiotic treatment alone. This was accompanied by reduced accumulation of immature myeloid cells in the lungs at the end of treatment and increased TNF-α protein levels throughout the treatment period. We demonstrate, in a mouse model of pulmonary TB, that immunotherapy consisting of three clinically approved drugs can improve the therapeutic efficacy of standard antibiotic treatment.

Role of Alternative Polyadenylation during Adipogenic Differentiation: An In Silico Approach

PubMed Central

Spangenberg, Lucía; Correa, Alejandro; Dallagiovanna, Bruno; Naya, Hugo

2013-01-01

Post-transcriptional regulation of stem cell differentiation is far from being completely understood. Changes in protein levels are not fully correlated with corresponding changes in mRNAs; the observed differences might be partially explained by post-transcriptional regulation mechanisms, such as alternative polyadenylation. This would involve changes in protein binding, transcript usage, miRNAs and other non-coding RNAs. In the present work we analyzed the distribution of alternative transcripts during adipogenic differentiation and the potential role of miRNAs in post-transcriptional regulation. Our in silico analysis suggests a modest, consistent, bias in 3′UTR lengths during differentiation enabling a fine-tuned transcript regulation via small non-coding RNAs. Including these effects in the analyses partially accounts for the observed discrepancies in relative abundance of protein and mRNA. PMID:24143171

Skin test performed with highly purified Mycobacterium tuberculosis recombinant protein triggers tuberculin shock in infected guinea pigs.

PubMed

Reece, Stephen T; Stride, Nicole; Ovendale, Pamela; Reed, Steven G; Campos-Neto, Antonio

2005-06-01

Tuberculin shock due to inoculation of Mycobacterium tuberculosis antigens in patients with tuberculosis is a serious syndrome originally described over 100 years ago by Robert Koch. Here, we present experimental evidence that a single M. tuberculosis recombinant protein, CFP-10, triggers this syndrome. Intradermal inoculation of CFP-10 elicits in M. tuberculosis-infected mice high levels of serum tumor necrosis factor alpha and causes tuberculin shock in infected guinea pigs characterized by hypothermia and death within 6 to 48 h after the antigen inoculation. Autopsies of these animals revealed intense polycythemia and hemorrhagic patches in the lung parenchyma, a pathological observation consistent with tuberculin shock. These results point to the possible occurrence of tuberculin shock in sensitive individuals inoculated with highly purified M. tuberculosis recombinant proteins as vaccine candidates or skin test reagents.

Defining Aggressive Prostate Cancer Using a 12-Gene Model1

PubMed Central

Riva, Alberto; Kim, Robert; Varambally, Sooryanarayana; He, Le; Kutok, Jeff; Aster, Jonathan C; Tang, Jeffery; Kuefer, Rainer; Hofer, Matthias D; Febbo, Phillip G; Chinnaiyan, Arul M; Rubin, Mark A

2006-01-01

Abstract The critical clinical question in prostate cancer research is: How do we develop means of distinguishing aggressive disease from indolent disease? Using a combination of proteomic and expression array data, we identified a set of 36 genes with concordant dysregulation of protein products that could be evaluated in situ by quantitative immunohistochemistry. Another five prostate cancer biomarkers were included using linear discriminant analysis, we determined that the optimal model used to predict prostate cancer progression consisted of 12 proteins. Using a separate patient population, transcriptional levels of the 12 genes encoding for these proteins predicted prostate-specific antigen failure in 79 men following surgery for clinically localized prostate cancer (P = .0015). This study demonstrates that cross-platform models can lead to predictive models with the possible advantage of being more robust through this selection process. PMID:16533427

Human L-ferritin deficiency is characterized by idiopathic generalized seizures and atypical restless leg syndrome

PubMed Central

Cozzi, Anna; Santambrogio, Paolo; Privitera, Daniela; Broccoli, Vania; Rotundo, Luisa Ida; Garavaglia, Barbara; Benz, Rudolf; Altamura, Sandro; Goede, Jeroen S.; Muckenthaler, Martina U.

2013-01-01

The ubiquitously expressed iron storage protein ferritin plays a central role in maintaining cellular iron homeostasis. Cytosolic ferritins are composed of heavy (H) and light (L) subunits that co-assemble into a hollow spherical shell with an internal cavity where iron is stored. The ferroxidase activity of the ferritin H chain is critical to store iron in its Fe3+ oxidation state, while the L chain shows iron nucleation properties. We describe a unique case of a 23-yr-old female patient affected by a homozygous loss of function mutation in the L-ferritin gene, idiopathic generalized seizures, and atypical restless leg syndrome (RLS). We show that L chain ferritin is undetectable in primary fibroblasts from the patient, and thus ferritin consists only of H chains. Increased iron incorporation into the FtH homopolymer leads to reduced cellular iron availability, diminished levels of cytosolic catalase, SOD1 protein levels, enhanced ROS production and higher levels of oxidized proteins. Importantly, key phenotypic features observed in fibroblasts are also mirrored in reprogrammed neurons from the patient’s fibroblasts. Our results demonstrate for the first time the pathophysiological consequences of L-ferritin deficiency in a human and help to define the concept for a new disease entity hallmarked by idiopathic generalized seizure and atypical RLS. PMID:23940258

T-kininogen inhibits kinin-mediated activation of ERK in endothelial cells.

PubMed

Leiva-Salcedo, Elias; Perez, Viviana; Acuña-Castillo, Claudio; Walter, Robin; Sierra, Felipe

2002-01-01

Serum levels of T-kininogen increase dramatically as rats approach the end of their lifespan. Stable expression of the protein in Balb/c 3T3 fibroblasts leads to a dramatic inhibition of cell proliferation, as well as inhibition of the ERK signaling pathway. T-kininogen is a potent inhibitor of cysteine proteinases, and we have described that the inhibition of ERK activity occurs, at least in part, via stabilization of the MAP kinase phosphatase, MKP-1. Since fibroblasts are not a physiological target of T-kininogen, we have now purified the protein from rat serum, and used it to assess the effect of T-kininogen on endothelial cells. Adding purified T-kininogen to EAhy 926 hybridoma cells resulted in inhibition of basal ERK activity levels, as estimated using appropriate anti-phospho ERK antibodies. Furthermore, exogenously added T-kininogen inhibited the activation of the ERK pathway induced by either bradykinin or T-kinin. We conclude that the age-related increase in hepatic T-kininogen gene expression and serum levels of the protein could have dramatic consequences on endothelial cell physiology, both under steady state conditions, and after activation by cell-specific stimuli. Our results are consistent with T-kininogen being an important modulator of the senescent phenotype in vivo.

Possible role of hypercoagulability in calciphylaxis: review of the literature.

PubMed

Harris, Ryan Jeffrey; Cropley, Thomas George

2011-02-01

The role of a hypercoagulable state in the pathogenesis of calciphylaxis has yet to be determined. We sought to find evidence of an association between hypercoagulability and calciphylaxis. We reviewed the primary literature for review articles, studies, and case reports that discussed or demonstrated a possible relationship between calciphylaxis and a hypercoagulable state. Review of the primary literature showed that in cases of calciphylaxis with reported levels of protein C and S, 38% of the patients had decreased protein C levels and 43% had decreased levels of protein S. From review of case reports, 3 cases of improvement of skin lesions with low molecular weight heparin treatment, and a fourth case of healing of skin lesions with tissue plasminogen activator treatment, were found. Calciphylaxis was also found in a patient with antiphospholipid antibody syndrome, and a patient with cryofibrinogenemia had clinical and histologic findings consistent with possible calciphylaxis. A limited number of reports were available for review. Our review of the literature found sufficient evidence to suggest a possible role of a hypercoagulable state in the pathogenesis of calciphylaxis. A prospective study with serial testing of all relevant clotting factors in patients with calciphylaxis is needed to more definitively establish this role. Copyright © 2009 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

Selection for low or high primary dormancy in Lolium rigidum Gaud seeds results in constitutive differences in stress protein expression and peroxidase activity

PubMed Central

Goggin, Danica E.; Powles, Stephen B.; Steadman, Kathryn J.

2011-01-01

Seed dormancy in wild Lolium rigidum Gaud (annual ryegrass) populations is highly variable and not well characterized at the biochemical level. To identify some of the determinants of dormancy level in these seeds, the proteomes of subpopulations selected for low and high levels of primary dormancy were compared by two-dimensional polyacrylamide gel electrophoresis of extracts from mature, dry seeds. High-dormancy seeds showed higher expression of small heat shock proteins, enolase, and glyoxalase I than the low-dormancy seeds. The functional relevance of these differences in protein expression was confirmed by the fact that high-dormancy seeds were more tolerant to high temperatures imposed at imbibition and had consistently higher glyoxalase I activity over 0–42 d dark stratification. Higher expression of a putative glutathione peroxidase in low-dormancy seeds was not accompanied by higher activity, but these seeds had a slightly more oxidized glutathione pool and higher total peroxidase activity. Overall, these biochemical and physiological differences suggest that L. rigidum seeds selected for low dormancy are more prepared for rapid germination via peroxidase-mediated cell wall weakening, whilst seeds selected for high dormancy are constitutively prepared to survive environmental stresses, even in the absence of stress during seed development. PMID:20974739

The inner CSF–brain barrier: developmentally controlled access to the brain via intercellular junctions

PubMed Central

Whish, Sophie; Dziegielewska, Katarzyna M.; Møllgård, Kjeld; Noor, Natassya M.; Liddelow, Shane A.; Habgood, Mark D.; Richardson, Samantha J.; Saunders, Norman R.

2015-01-01

In the adult the interface between the cerebrospinal fluid and the brain is lined by the ependymal cells, which are joined by gap junctions. These intercellular connections do not provide a diffusional restrain between the two compartments. However, during development this interface, initially consisting of neuroepithelial cells and later radial glial cells, is characterized by “strap” junctions, which limit the exchange of different sized molecules between cerebrospinal fluid and the brain parenchyma. Here we provide a systematic study of permeability properties of this inner cerebrospinal fluid-brain barrier during mouse development from embryonic day, E17 until adult. Results show that at fetal stages exchange across this barrier is restricted to the smallest molecules (286Da) and the diffusional restraint is progressively removed as the brain develops. By postnatal day P20, molecules the size of plasma proteins (70 kDa) diffuse freely. Transcriptomic analysis of junctional proteins present in the cerebrospinal fluid-brain interface showed expression of adherens junctional proteins, actins, cadherins and catenins changing in a development manner consistent with the observed changes in the permeability studies. Gap junction proteins were only identified in the adult as was claudin-11. Immunohistochemistry was used to localize at the cellular level some of the adherens junctional proteins of genes identified from transcriptomic analysis. N-cadherin, β - and α-catenin immunoreactivity was detected outlining the inner CSF-brain interface from E16; most of these markers were not present in the adult ependyma. Claudin-5 was present in the apical-most part of radial glial cells and in endothelial cells in embryos, but only in endothelial cells including plexus endothelial cells in adults. Claudin-11 was only immunopositive in the adult, consistent with results obtained from transcriptomic analysis. These results provide information about physiological, molecular and morphological-related permeability changes occurring at the inner cerebrospinal fluid-brain barrier during brain development. PMID:25729345

Unraveling patterns of site-to-site synonymous rates variation and associated gene properties of protein domains and families.

PubMed

Dimitrieva, Slavica; Anisimova, Maria

2014-01-01

In protein-coding genes, synonymous mutations are often thought not to affect fitness and therefore are not subject to natural selection. Yet increasingly, cases of non-neutral evolution at certain synonymous sites were reported over the last decade. To evaluate the extent and the nature of site-specific selection on synonymous codons, we computed the site-to-site synonymous rate variation (SRV) and identified gene properties that make SRV more likely in a large database of protein-coding gene families and protein domains. To our knowledge, this is the first study that explores the determinants and patterns of the SRV in real data. We show that the SRV is widespread in the evolution of protein-coding sequences, putting in doubt the validity of the synonymous rate as a standard neutral proxy. While protein domains rarely undergo adaptive evolution, the SRV appears to play important role in optimizing the domain function at the level of DNA. In contrast, protein families are more likely to evolve by positive selection, but are less likely to exhibit SRV. Stronger SRV was detected in genes with stronger codon bias and tRNA reusage, those coding for proteins with larger number of interactions or forming larger number of structures, located in intracellular components and those involved in typically conserved complex processes and functions. Genes with extreme SRV show higher expression levels in nearly all tissues. This indicates that codon bias in a gene, which often correlates with gene expression, may often be a site-specific phenomenon regulating the speed of translation along the sequence, consistent with the co-translational folding hypothesis. Strikingly, genes with SRV were strongly overrepresented for metabolic pathways and those associated with several genetic diseases, particularly cancers and diabetes.

Changes in the leaf proteome profile of Withania somnifera (L.) Dunal in response to Alternaria alternata infection

PubMed Central

Singh, Varinder; Singh, Baldev; Joshi, Robin; Jaju, Puneet

2017-01-01

Withania somnifera is a high value medicinal plant which is used against large number of ailments. The medicinal properties of the plant attributes to a wide array of important secondary metabolites. The plant is predominantly infected with leaf spot pathogen Alternaria alternata, which leads to substantial biodeterioration of pharmaceutically important metabolites. To develop an effective strategy to combat this disease, proteomics based approach could be useful. Hence, in the present study, three different protein extraction methods tris-buffer based, phenol based and trichloroacetic acid-acetone (TCA-acetone) based method were comparatively evaluated for two-dimensional electrophoresis (2-DE) analysis of W. somnifera. TCA-acetone method was found to be most effective and was further used to identify differentially expressed proteins in response to fungal infection. Thirty-eight differentially expressed proteins were identified by matrix assisted laser desorption/ionization time of flight-mass spectrometry (MALDI TOF/TOF MS/MS). The known proteins were categorized into eight different groups based on their function and maximum proteins belonged to energy and metabolism, cell structure, stress and defense and RNA/DNA categories. Differential expression of some key proteins were also crosschecked at transcriptomic level by using qRT-PCR and were found to be consistent with the 2-DE data. These outcomes enable us to evaluate modifications that take place at the proteomic level during a compatible host pathogen interaction. The comparative proteome analysis conducted in this paper revealed the involvement of many key proteins in the process of pathogenesis and further investigation of these identified proteins could assist in the discovery of new strategies for the development of pathogen resistance in the plant. PMID:28575108

The PNPLA3 variant associated with fatty liver disease (I148M) accumulates on lipid droplets by evading ubiquitylation.

PubMed

BasuRay, Soumik; Smagris, Eriks; Cohen, Jonathan C; Hobbs, Helen H

2017-10-01

A sequence variation (I148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) is strongly associated with fatty liver disease, but the underlying mechanism remains obscure. In this study, we used knock-in (KI) mice (Pnpla3 148M/M ) to examine the mechanism responsible for accumulation of triglyceride (TG) and PNPLA3 in hepatic lipid droplets (LDs). No differences were found between Pnpla3 148M/M and Pnpla3 +/+ mice in hepatic TG synthesis, utilization, or secretion. These results are consistent with TG accumulation in the Pnpla3 148M/M mice being caused by impaired TG mobilization from LDs. Sucrose feeding, which is required to elicit fatty liver in KI mice, led to a much larger and more persistent increase in PNPLA3 protein in the KI mice than in wild-type (WT) mice. Inhibition of the proteasome (bortezomib), but not macroautophagy (3-methyladenine), markedly increased PNPLA3 levels in WT mice, coincident with the appearance of ubiquitylated forms of the protein. Bortezomib did not increase PNPLA3 levels in Pnpla3 148M/M mice, and only trace amounts of ubiquitylated PNPLA3 were seen in these animals. These results are consistent with the notion that the 148M variant disrupts ubiquitylation and proteasomal degradation of PNPLA3, resulting in accumulation of PNPLA3-148M and impaired mobilization of TG from LDs. (Hepatology 2017;66:1111-1124). © 2017 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.

Cyanogen Metabolism in Cassava Roots: Impact on Protein Synthesis and Root Development.

PubMed

Zidenga, Tawanda; Siritunga, Dimuth; Sayre, Richard T

2017-01-01

Cassava ( Manihot esculenta Crantz), a staple crop for millions of sub-Saharan Africans, contains high levels of cyanogenic glycosides which protect it against herbivory. However, cyanogens have also been proposed to play a role in nitrogen transport from leaves to roots. Consistent with this hypothesis, analyses of the distribution and activities of enzymes involved in cyanide metabolism provides evidence for cyanide assimilation, derived from linamarin, into amino acids in cassava roots. Both β-cyanoalanine synthase (CAS) and nitrilase (NIT), two enzymes involved in cyanide assimilation to produce asparagine, were observed to have higher activities in roots compared to leaves, consistent with their proposed role in reduced nitrogen assimilation. In addition, rhodanese activity was not detected in cassava roots, indicating that this competing means for cyanide metabolism was not a factor in cyanide detoxification. In contrast, leaves had sufficient rhodanese activity to compete with cyanide assimilation into amino acids. Using transgenic low cyanogen plants, it was shown that reducing root cyanogen levels is associated with elevated root nitrate reductase activity, presumably to compensate for the loss of reduced nitrogen from cyanogens. Finally, we overexpressed Arabidopsis CAS and NIT4 genes in cassava roots to study the feasibility of enhancing root cyanide assimilation into protein. Optimal overexpression of CAS and NIT4 resulted in up to a 50% increase in root total amino acids and a 9% increase in root protein accumulation. However, plant growth and morphology was altered in plants overexpressing these enzymes, demonstrating a complex interaction between cyanide metabolism and hormonal regulation of plant growth.

Cyanogen Metabolism in Cassava Roots: Impact on Protein Synthesis and Root Development

PubMed Central

Zidenga, Tawanda; Siritunga, Dimuth; Sayre, Richard T.

2017-01-01

Cassava (Manihot esculenta Crantz), a staple crop for millions of sub-Saharan Africans, contains high levels of cyanogenic glycosides which protect it against herbivory. However, cyanogens have also been proposed to play a role in nitrogen transport from leaves to roots. Consistent with this hypothesis, analyses of the distribution and activities of enzymes involved in cyanide metabolism provides evidence for cyanide assimilation, derived from linamarin, into amino acids in cassava roots. Both β-cyanoalanine synthase (CAS) and nitrilase (NIT), two enzymes involved in cyanide assimilation to produce asparagine, were observed to have higher activities in roots compared to leaves, consistent with their proposed role in reduced nitrogen assimilation. In addition, rhodanese activity was not detected in cassava roots, indicating that this competing means for cyanide metabolism was not a factor in cyanide detoxification. In contrast, leaves had sufficient rhodanese activity to compete with cyanide assimilation into amino acids. Using transgenic low cyanogen plants, it was shown that reducing root cyanogen levels is associated with elevated root nitrate reductase activity, presumably to compensate for the loss of reduced nitrogen from cyanogens. Finally, we overexpressed Arabidopsis CAS and NIT4 genes in cassava roots to study the feasibility of enhancing root cyanide assimilation into protein. Optimal overexpression of CAS and NIT4 resulted in up to a 50% increase in root total amino acids and a 9% increase in root protein accumulation. However, plant growth and morphology was altered in plants overexpressing these enzymes, demonstrating a complex interaction between cyanide metabolism and hormonal regulation of plant growth. PMID:28286506

The role of arginine metabolic pathway during embryogenesis and germination in maritime pine (Pinus pinaster Ait.).

PubMed

Llebrés, María-Teresa; Pascual, María-Belén; Debille, Sandrine; Trontin, Jean-François; Harvengt, Luc; Avila, Concepción; Cánovas, Francisco M

2018-03-01

Vegetative propagation through somatic embryogenesis is critical in conifer biotechnology towards multivarietal forestry that uses elite varieties to cope with environmental and socio-economic issues. An important and still sub-optimal process during in vitro maturation of somatic embryos (SE) is the biosynthesis and deposition of storage proteins, which are rich in amino acids with high nitrogen (N) content, such as arginine. Mobilization of these N-rich proteins is essential for the germination and production of vigorous somatic seedlings. Somatic embryos accumulate lower levels of N reserves than zygotic embryos (ZE) at a similar stage of development. To understand the molecular basis for this difference, the arginine metabolic pathway has been characterized in maritime pine (Pinus pinaster Ait.). The genes involved in arginine metabolism have been identified and GFP-fusion constructs were used to locate the enzymes in different cellular compartments and clarify their metabolic roles during embryogenesis and germination. Analysis of gene expression during somatic embryo maturation revealed high levels of transcripts for genes involved in the biosynthesis and metabolic utilization of arginine. By contrast, enhanced expression levels were only observed during the last stages of maturation and germination of ZE, consistent with the adequate accumulation and mobilization of protein reserves. These results suggest that arginine metabolism is unbalanced in SE (simultaneous biosynthesis and degradation of arginine) and could explain the lower accumulation of storage proteins observed during the late stages of somatic embryogenesis.

The Heterotrimeric G-Protein Subunits GNG-1 and GNB-1 Form a Gβγ Dimer Required for Normal Female Fertility, Asexual Development, and Gα Protein Levels in Neurospora crassa

PubMed Central

Krystofova, Svetlana; Borkovich, Katherine A.

2005-01-01

We have identified a gene encoding a heterotrimeric G protein γ subunit, gng-1, from the filamentous fungus Neurospora crassa. gng-1 possesses a gene structure similar to that of mammalian Gγ genes, consisting of three exons and two introns, with introns present in both the open reading frame and 5′-untranslated region. The GNG-1 amino acid sequence displays high identity to predicted Gγ subunits from other filamentous fungi, including Giberella zeae, Cryphonectria parasitica, Trichoderma harzianum, and Magnaporthe grisea. Deletion of gng-1 leads to developmental defects similar to those previously characterized for Δgnb-1 (Gβ) mutants. Δgng-1, Δgnb-1, and Δgng-1 Δgnb-1 strains conidiate inappropriately in submerged cultures and are female sterile, producing aberrant female reproductive structures. Similar to previous results obtained with Δgnb-1 mutants, loss of gng-1 negatively influences levels of Gα proteins (GNA-1, GNA-2, and GNA-3) in plasma membrane fractions isolated from various tissues of N. crassa and leads to a significant reduction in the amount of intracellular cyclic AMP. In addition, we show that GNB-1 is essential for maintenance of normal steady-state levels of GNG-1, suggesting a functional interaction between GNB-1 and GNG-1. Direct evidence for a physical association between GNB-1 and GNG-1 in vivo was provided by coimmunoprecipitation. PMID:15701799

Time- and dose-related interactions between glucocorticoid and cyclic adenosine 3',5'-monophosphate on CCAAT/enhancer-binding protein-dependent insulin-like growth factor I expression by osteoblasts

NASA Technical Reports Server (NTRS)

McCarthy, T. L.; Ji, C.; Chen, Y.; Kim, K.; Centrella, M.

2000-01-01

Glucocorticoid has complex effects on osteoblasts. Several of these changes appear to be related to steroid concentration, duration of exposure, or specific effects on growth factor expression or activity within bone. One important bone growth factor, insulin-like growth factor I (IGF-I), is induced in osteoblasts by hormones such as PGE2 that increase intracellular cAMP levels. In this way, PGE2 activates transcription factor CCAAT/enhancer-binding protein-delta (C/EBPdelta) and enhances its binding to a specific control element found in exon 1 in the IGF-I gene. Our current studies show that preexposure to glucocorticoid enhanced C/EBPdelta and C/EBPbeta expression by osteoblasts and thereby potentiated IGF-I gene promoter activation in response to PGE2. Importantly, this directly contrasts with inhibitory effects on IGF-I expression that result from sustained or pharmacologically high levels of glucocorticoid exposure. Consistent with the stimulatory effect of IGF-I on bone protein synthesis, pretreatment with glucocorticoid sensitized osteoblasts to PGE2, and in this context significantly enhanced new collagen and noncollagen protein synthesis. Therefore, pharmacological levels of glucocorticoid may reduce IGF-I expression by osteoblasts and cause osteopenic disease, whereas physiological transient increases in glucocorticoid may permit or amplify the effectiveness of hormones that regulate skeletal tissue integrity. These events appear to converge on the important role of C/EBPdelta and C/EBPbeta on IGF-I expression by osteoblasts.

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells

PubMed Central

Sanchez-Lopez, E; Zimmerman, T; Gomez del Pulgar, T; Moyer, M P; Lacal Sanjuan, J C; Cebrian, A

2013-01-01

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction. PMID:24287694

Peroxiredoxin I protein, a potential biomarker of hydronephrosis in fetal mice exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

PubMed

Liu, Mingxue; Liu, Jing; Liu, Xing; Wei, Guanghui

2014-06-01

In previous studies, we established an animal model of human congenital hydronephrosis with exposure of developing mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but the etiopathogenesis is not entirely clear. The present study was to identify the changes that may be involved in the etiology at the protein level. C57BL/6J mice fetuses were treated with TCDD. Comparative proteomic analysis was adopted to identify the proteins associated with hydronephrosis induced by TCDD. Two-dimensional electrophoresis display revealed that 19 protein spots were differentially expressed in the upper urinary tract tissues in fetal mice after exposure to TCDD. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) identified 12 up-regulated proteins: peroxiredoxin I (Prx I), cadherin 6, gamma-actin, radixin, desmin, type II transforming growth factor-beta receptor, chromogranin B, serum albumin precursor, transferrin, hypothetical protein LOC70984, lipk protein, and zinc finger protein 336. Histochemical staining indicated that Prx I protein was positively expressed in the ureteric epithelium in the treated group, and not in the control group, which is consistent with MALDI-TOF-MS. Prx I protein may be a potential biomarker or responsive protein of hydronephrosis in fetal mice induced by TCDD. Copyright © 2013 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

Human Spermatozoa Contain Multiple Targets for Protein S-Nitrosylation: An Alternative Mechanism of the Modulation of Sperm Function by Nitric Oxide?

PubMed Central

Lefièvre, Linda; Chen, Yongjian; Conner, Sarah J; Scott, Joanna L; Publicover, Steve J; Ford, W Christopher L; Barratt, Christopher LR

2009-01-01

Nitric oxide (NO) enhances human sperm motility and capacitation associated with increased protein phosphorylation. NO activates soluble guanylyl cyclase, but can also modify protein function covalently via S-nitrosylation of cysteine. Remarkably, this mechanism remains unexplored in sperm although they depend on post-translational protein modification to achieve changes in function required for fertilisation. Our objective was to identify targets for S-nitrosylation in human sperm. Spermatozoa were incubated with NO donors and S-nitrosylated proteins were identified using the biotin switch assay and a proteomic approach using tandem mass spectrometry. 240 S-nitrosylated proteins were detected in sperm incubated with S-nitrosoglutathione. Minimal levels were observed in glutathione or untreated samples. Proteins identified consistently based on multiple peptides included established targets for S-nitrosylation in other cells e.g. tubulin,, glutathione-S-transferase and heat shock proteins but also novel targets including A-kinase anchoring protein (AKAP) types 3 and 4, voltage-dependent anion-selective channel protein 3 and semenogelin 1 and 2. In situ localisation revealed S-nitrosylated targets on the post-acrosomal region of the head and throughout the flagellum. Potential targets for S-nitrosylation in human sperm include physiologically significant proteins not previously reported in other cells. Their identification will provide novel insight into the mechanism of action of NO in spermatozoa. PMID:17683036

EAST Organizes Drosophila Insulator Proteins in the Interchromosomal Nuclear Compartment and Modulates CP190 Binding to Chromatin

PubMed Central

Golovnin, Anton; Melnikova, Larisa; Shapovalov, Igor; Kostyuchenko, Margarita; Georgiev, Pavel

2015-01-01

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization. PMID:26489095

Variance in total levels of phospholipase C zeta (PLC-ζ) in human sperm may limit the applicability of quantitative immunofluorescent analysis as a diagnostic indicator of oocyte activation capability.

PubMed

Kashir, Junaid; Jones, Celine; Mounce, Ginny; Ramadan, Walaa M; Lemmon, Bernadette; Heindryckx, Bjorn; de Sutter, Petra; Parrington, John; Turner, Karen; Child, Tim; McVeigh, Enda; Coward, Kevin

2013-01-01

To examine whether similar levels of phospholipase C zeta (PLC-ζ) protein are present in sperm from men whose ejaculates resulted in normal oocyte activation, and to examine whether a predominant pattern of PLC-ζ localization is linked to normal oocyte activation ability. Laboratory study. University laboratory. Control subjects (men with proven oocyte activation capacity; n = 16) and men whose sperm resulted in recurrent intracytoplasmic sperm injection failure (oocyte activation deficient [OAD]; n = 5). Quantitative immunofluorescent analysis of PLC-ζ protein in human sperm. Total levels of PLC-ζ fluorescence, proportions of sperm exhibiting PLC-ζ immunoreactivity, and proportions of PLC-ζ localization patterns in sperm from control and OAD men. Sperm from control subjects presented a significantly higher proportion of sperm exhibiting PLC-ζ immunofluorescence compared with infertile men diagnosed with OAD (82.6% and 27.4%, respectively). Total levels of PLC-ζ in sperm from individual control and OAD patients exhibited significant variance, with sperm from 10 out of 16 (62.5%) exhibiting levels similar to OAD samples. Predominant PLC-ζ localization patterns varied between control and OAD samples with no predictable or consistent pattern. The results indicate that sperm from control men exhibited significant variance in total levels of PLC-ζ protein, as well as significant variance in the predominant localization pattern. Such variance may hinder the diagnostic application of quantitative PLC-ζ immunofluorescent analysis. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Branched-chain amino acids supplementation protects streptozotocin-induced insulin secretion and the correlated mechanism.

PubMed

Lu, Ming; Zhang, Xiujuan; Zheng, Dongmei; Jiang, Xiuyun; Chen, Qing

2015-01-01

Significant evidence demonstrates that oxidative stress can impair insulin secretion and contribute to the development of type 2 diabetes. Branched-chain amino acids (BCAAs) are reported to be positively related to insulin secretion. This study aimed to determine how oxidative stress affects the function of islets and whether BCAAs can ameliorate the oxidative stress, and accompanying c-jun N-terminal kinase (JNK), protein kinase D1 (PKD1), and pancreatic/duodenal homeobox-1 (PDX-1) changes induced by streptozotocin (STZ). Plasma glucose, plasma insulin, and JNK, PKD1 and PDX-1 mRNA and protein expression were measured in rats treated with STZ and BCAAs. The glucose level in STZ-induced diabetic rats was much higher than that in control animals, and the elevated plasma glucose level in diabetic rats could be significantly inhibited by BCAAs treatment. Consistent with the change in glucose levels, the levels of insulin were also affected by BCAAs treatment. The mRNA and protein expression of JNK, PDX-1, and PKD1 were significantly altered in diabetic rats compared with the control group (P<0.01) and treatment with a low dose of BCAA reversed these changes in those above markers significantly (P<0.01). The present study demonstrated that STZ-induced oxidative stress could reduce serum insulin levels and alter the JNK, PDX-1, and PKD1 expression. BCAAs restored the levels of serum insulin reversed changes in JNK, PDX-1, and PKD1 expression. © 2014 International Union of Biochemistry and Molecular Biology.

Markers of apoptosis induction and proliferation in the orbitofrontal cortex in alcohol dependence

PubMed Central

Whittom, Angela; Villarreal, Ashley; Soni, Madhav; Owusu-Duku, Beverly; Meshram, Ashish; Rajkowska, Grazyna; Stockmeier, Craig A.; Miguel-Hidalgo, Jose J.

2014-01-01

Background Alcohol-dependent (ALC) subjects exhibit glial and neuronal pathology in the prefrontal cortex (PFC). However, in many patients, neurophysiological disturbances are not associated with catastrophic cell depletion despite prolonged alcohol abuse. It is still unclear how some relevant markers of a cell’s propensity to degenerate or proliferate are changed in the PFC of ALC subjects without major neurological disorders. Methods Levels of pro-apoptotic caspase 8 (C8), X-linked inhibitor of apoptosis protein (XIAP), direct IAP binding protein with low pI (DIABLO), proliferating cell nuclear antigen (PCNA), and density of cells immunoreactive (-IR) for proliferation marker Ki-67 were measured postmortem in the left orbitofrontal cortex (OFC) of 29 subjects with alcohol dependence and 23 non-psychiatric comparison subjects. Results ALC subjects had significantly higher levels of the 14kDa C8 fragment (C8-14), an indicator of C8 activation. However, there was no change in the levels of DIABLO, XIAP or in the DIABLO/XIAP ratio. PCNA protein level and density of Ki-67-IR cells were not significantly changed in alcoholics, although PCNA levels were increased in older ALC subjects as compared to controls. Conclusions Significant increase of a C8 activation indicator was found in alcoholism, but without significant changes in XIAP level, DIABLO/XIAP ratio, or Ki-67 labeling. These results would help to explain the absence of catastrophic cell loss in the PFC of many alcohol dependent subjects, while still being consistent with an alcoholism-related vulnerability to slow decline in glial cells and neurons in the OFC of alcoholics. PMID:25421516

A Chronic Obstructive Pulmonary Disease Susceptibility Gene, FAM13A, Regulates Protein Stability of β-Catenin.

PubMed

Jiang, Zhiqiang; Lao, Taotao; Qiu, Weiliang; Polverino, Francesca; Gupta, Kushagra; Guo, Feng; Mancini, John D; Naing, Zun Zar Chi; Cho, Michael H; Castaldi, Peter J; Sun, Yang; Yu, Jane; Laucho-Contreras, Maria E; Kobzik, Lester; Raby, Benjamin A; Choi, Augustine M K; Perrella, Mark A; Owen, Caroline A; Silverman, Edwin K; Zhou, Xiaobo

2016-07-15

A genetic locus within the FAM13A gene has been consistently associated with chronic obstructive pulmonary disease (COPD) in genome-wide association studies. However, the mechanisms by which FAM13A contributes to COPD susceptibility are unknown. To determine the biologic function of FAM13A in human COPD and murine COPD models and discover the molecular mechanism by which FAM13A influences COPD susceptibility. Fam13a null mice (Fam13a(-/-)) were generated and exposed to cigarette smoke. The lung inflammatory response and airspace size were assessed in Fam13a(-/-) and Fam13a(+/+) littermate control mice. Cellular localization of FAM13A protein and mRNA levels of FAM13A in COPD lungs were assessed using immunofluorescence, Western blotting, and reverse transcriptase-polymerase chain reaction, respectively. Immunoprecipitation followed by mass spectrometry identified cellular proteins that interact with FAM13A to reveal insights on FAM13A's function. In murine and human lungs, FAM13A is expressed in airway and alveolar type II epithelial cells and macrophages. Fam13a null mice (Fam13a(-/-)) were resistant to chronic cigarette smoke-induced emphysema compared with Fam13a(+/+) mice. In vitro, FAM13A interacts with protein phosphatase 2A and recruits protein phosphatase 2A with glycogen synthase kinase 3β and β-catenin, inducing β-catenin degradation. Fam13a(-/-) mice were also resistant to elastase-induced emphysema, and this resistance was reversed by coadministration of a β-catenin inhibitor, suggesting that FAM13A could increase the susceptibility of mice to emphysema development by inhibiting β-catenin signaling. Moreover, human COPD lungs had decreased protein levels of β-catenin and increased protein levels of FAM13A. We show that FAM13A may influence COPD susceptibility by promoting β-catenin degradation.

Highly efficient strategy for the heterologous expression and purification of soluble Cowpea chlorotic mottle virus capsid protein and in vitro pH-dependent assembly of virus-like particles.

PubMed

Díaz-Valle, Armando; García-Salcedo, Yardena M; Chávez-Calvillo, Gabriela; Silva-Rosales, Laura; Carrillo-Tripp, Mauricio

2015-12-01

Obtaining pure and soluble viral capsid proteins (CPs) has been a major challenge in the fields of science and technology in recent decades. In many cases, the CPs can self-assemble in the absence of a viral genome, resulting in non-infectious, empty virus-like particles (VLPs) which can be safely handled. The use of VLPs has found great potential in biotechnology and health purposes. In addition, VLPs are a good model system to study protein-protein interactions at the molecular level. In this work, an optimized strategy for the heterologous expression of the Cowpea chlorotic mottle virus (CCMV) CP based in Escherichia coli is described. The method is efficient, inexpensive and it consistently produces higher yields and greater purity levels than those reported so far. Additionally, one of the main advantages of this method is the prevention of the formation of inclusion bodies, thus allowing to directly obtain high amounts of the CP in a soluble and functionally active state with the capacity to readily form VLPs in vitro. The CCMV CP self-assembly pH dependence was also investigated, providing guidelines to easily modulate the process. Copyright © 2015 Elsevier B.V. All rights reserved.

Networks of blood proteins in the neuroimmunology of schizophrenia.

PubMed

Jeffries, Clark D; Perkins, Diana O; Fournier, Margot; Do, Kim Q; Cuenod, Michel; Khadimallah, Ines; Domenici, Enrico; Addington, Jean; Bearden, Carrie E; Cadenhead, Kristin S; Cannon, Tyrone D; Cornblatt, Barbara A; Mathalon, Daniel H; McGlashan, Thomas H; Seidman, Larry J; Tsuang, Ming; Walker, Elaine F; Woods, Scott W

2018-06-06

Levels of certain circulating cytokines and related immune system molecules are consistently altered in schizophrenia and related disorders. In addition to absolute analyte levels, we sought analytes in correlation networks that could be prognostic. We analyzed baseline blood plasma samples with a Luminex platform from 72 subjects meeting criteria for a psychosis clinical high-risk syndrome; 32 subjects converted to a diagnosis of psychotic disorder within two years while 40 other subjects did not. Another comparison group included 35 unaffected subjects. Assays of 141 analytes passed early quality control. We then used an unweighted co-expression network analysis to identify highly correlated modules in each group. Overall, there was a striking loss of network complexity going from unaffected subjects to nonconverters and thence to converters (applying standard, graph-theoretic metrics). Graph differences were largely driven by proteins regulating tissue remodeling (e.g. blood-brain barrier). In more detail, certain sets of antithetical proteins were highly correlated in unaffected subjects (e.g. SERPINE1 vs MMP9), as expected in homeostasis. However, for particular protein pairs this trend was reversed in converters (e.g. SERPINE1 vs TIMP1, being synthetical inhibitors of remodeling of extracellular matrix and vasculature). Thus, some correlation signals strongly predict impending conversion to a psychotic disorder and directly suggest pharmaceutical targets.

[Clustered regularly interspaced short palindromic repeat associated protein genes cas1 and cas2 in Shigella].

PubMed

Xue, Zerun; Wang, Yingfang; Duan, Guangcai; Wang, Pengfei; Wang, Linlin; Guo, Xiangjiao; Xi, Yuanlin

2014-05-01

To detect the distribution of clustered regularly interspaced short palindromic repeat (CRISPR) associated protein genes cas1 and cas2 in Shigella and to understand the characteristics of CRISPR with relationship between CRISPR and related characteristics on drug resistance. CRISPR associated protein genes cas1 and cas2 in Shigella were detected by PCR, with its products sequenced and compared. The CRISPR-associated protein genes cas1 and cas2 were found in all the 196 Shigella isolates which were isolated at different times and locations in China. Consistencies showed through related sequencing appeared as follows: cas2, cas1 (a) and cas1 (b) were 96.44%, 97.61% and 96.97%, respectively. There were two mutations including 3177129 site(C→G)and 3177126 site (G→C) of cas1 (b) gene in 2003135 strain which were not found in the corresponding sites of Z23 and 2008113. showed that in terms of both susceptibility and antibiotic-resistance, strain 2003135 was stronger than Z23 and 2008113. CRISPR system widely existed in Shigella, with the level of drug resistance in cas1 (b) gene mutant strains higher than in wild strains. Cas1 (b) gene mutation might be one of the reasons causing the different levels of resistance.

Age-related effect of aerobic exercise training on antioxidant and oxidative markers in the liver challenged by doxorubicin in rats.

PubMed

Ahmadian, Mehdi; Dabidi Roshan, Valiollah; Leicht, Anthony S

2018-05-16

The aims of the current study were to investigate the oxidant and antioxidant status of liver tissue challenged by doxorubicin and to examine the possible protective effects of aerobic exercise on doxorubicin-induced oxidative stress. Seventy-two rats were divided into three age groups (Young, Adult, and Elderly) with three treatment subgroups consisting of eight rats per age group: doxorubicin, aerobic exercise + doxorubicin, and aerobic exercise + saline. The experimental groups performed regular treadmill running for 3 weeks. Doxorubicin was administered by i.p. injection at a dosage of 20 mg kg -1 while the aerobic exercise + saline group received saline of a comparable volume. Heat shock protein 70, malondialdehyde, glutathione peroxidase, and protein carbonyl were determined from the liver homogenates following the intervention period. Treatment with doxorubicin induced hepatotoxicity in all groups with lower values of oxidative stress in young compared with the older groups. The inclusion of aerobic exercise training significantly increased heat shock protein 70 and antioxidant enzyme levels (glutathione peroxidase) whereas it decreased oxidative stress biomarkers (malondialdehyde and protein carbonyl) for all age groups. These results suggest that aerobic exercise training may be a potential, non-drug strategy to modulate doxorubicin-induced hepatotoxicity through its positive impact on antioxidant levels and oxidative stress biomarkers.

The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr

PubMed Central

Castiel, Asher; Danieli, Michal Mark; David, Ahuvit; Moshkovitz, Sharon; Aplan, Peter D.; Kirsch, Ilan R.; Brandeis, Michael; Krämer, Alwin; Izraeli, Shai

2011-01-01

Stil (Sil, SCL/TAL1 interrupting locus) is a cytosolic and centrosomal protein expressed in proliferating cells that is required for mouse and zebrafish neural development and is mutated in familial microcephaly. Recently the Drosophila melanogaster ortholog of Stil was found to be important for centriole duplication. Consistent with this finding, we report here that mouse embryonic fibroblasts lacking Stil are characterized by slow growth, low mitotic index and absence of clear centrosomes. We hypothesized that Stil regulates mitosis through the tumor suppressor Chfr, an E3 ligase that blocks mitotic entry in response to mitotic stress. Mouse fibroblasts lacking Stil by genomic or RNA interference approaches, as well as E9.5 Stil−/− embryos, express high levels of the Chfr protein and reduced levels of the Chfr substrate Plk1. Exogenous expression of Stil, knockdown of Chfr or overexpression of Plk1 reverse the abnormal mitotic phenotypes of fibroblasts lacking Stil. We further demonstrate that Stil increases Chfr auto-ubiquitination and reduces its protein stability. Thus, Stil is required for centrosome organization, entry into mitosis and cell proliferation, and these functions are at least partially mediated by Chfr and its targets. This is the first identification of a negative regulator of the Chfr mitotic checkpoint. PMID:21245198

Quantum Electron Tunneling in Respiratory Complex I1

PubMed Central

Hayashi, Tomoyuki; Stuchebrukhov, Alexei A.

2014-01-01

We have simulated the atomistic details of electronic wiring of all Fe/S clusters in complex I, a key enzyme in the respiratory electron transport chain. The tunneling current theory of many-electron systems is applied to the broken-symmetry (BS) states of the protein at the ZINDO level. One-electron tunneling approximation is found to hold in electron tunneling between the anti-ferromagnetic binuclear and tetranuclear Fe/S clusters with moderate induced polarization of the core electrons. Calculated tunneling energy is about 3 eV higher than Fermi level in the band gap of the protein, which supports that the mechanism of electron transfer is quantum mechanical tunneling, as in the rest of electron transport chain. Resulting electron tunneling pathways consist of up to three key contributing protein residues between neighboring Fe/S clusters. A distinct signature of the wave properties of electrons is observed as quantum interferences when multiple tunneling pathways exist. In N6a-N6b, electron tunnels along different pathways depending on the involved BS states, suggesting possible fluctuations of the tunneling pathways driven by the local protein environment. The calculated distance dependence of the electron transfer rates with internal water molecules included are in good agreement with a reported phenomenological relation. PMID:21495666

Minimum requirements for the function of eukaryotic translation initiation factor 2.

PubMed Central

Erickson, F L; Nika, J; Rippel, S; Hannig, E M

2001-01-01

Eukaryotic translation initiation factor 2 (eIF2) is a G protein heterotrimer required for GTP-dependent delivery of initiator tRNA to the ribosome. eIF2B, the nucleotide exchange factor for eIF2, is a heteropentamer that, in yeast, is encoded by four essential genes and one nonessential gene. We found that increased levels of wild-type eIF2, in the presence of sufficient levels of initiator tRNA, overcome the requirement for eIF2B in vivo. Consistent with bypassing eIF2B, these conditions also suppress the lethal effect of overexpressing the mammalian tumor suppressor PKR, an eIF2alpha kinase. The effects described are further enhanced in the presence of a mutation in the G protein (gamma) subunit of eIF2, gcd11-K250R, which mimics the function of eIF2B in vitro. Interestingly, the same conditions that bypass eIF2B also overcome the requirement for the normally essential eIF2alpha structural gene (SUI2). Our results suggest that the eIF2betagamma complex is capable of carrying out the essential function(s) of eIF2 in the absence of eIF2alpha and eIF2B and are consistent with the idea that the latter function primarily to regulate the level of eIF2.GTP.Met-tRNA(i)(Met) ternary complexes in vivo. PMID:11333223

Cys34 Adductomes Differ between Patients with Chronic Lung or Heart Disease and Healthy Controls in Central London.

PubMed

Liu, Sa; Grigoryan, Hasmik; Edmands, William M B; Dagnino, Sonia; Sinharay, Rudy; Cullinan, Paul; Collins, Peter; Chung, Kian Fan; Barratt, Benjamin; Kelly, Frank J; Vineis, Paolo; Rappaport, Stephen M

2018-02-20

Oxidative stress generates reactive species that modify proteins, deplete antioxidant defenses, and contribute to chronic obstructive pulmonary disease (COPD) and ischemic heart disease (IHD). To determine whether protein modifications differ between COPD or IHD patients and healthy subjects, we performed untargeted analysis of adducts at the Cys34 locus of human serum albumin (HSA). Biospecimens were obtained from nonsmoking participants from London, U.K., including healthy subjects (n = 20) and patients with COPD (n = 20) or IHD (n = 10). Serum samples were digested with trypsin and analyzed by liquid chromatography-high resolution mass spectrometry. Effects of air pollution on adduct levels were also investigated based on estimated residential exposures to PM 2.5 , O 3 and NO 2 . For the 39 adducts with sufficient data, levels were essentially identical in blood samples collected from the same subjects on two consecutive days, consistent with the 28 day residence time of HSA. Multivariate linear regression revealed 21 significant associations, mainly with the underlying diseases but also with air-pollution exposures (p-value < 0.05). Interestingly, most of the associations indicated that adduct levels decreased with the presence of disease or increased pollutant concentrations. Negative associations of COPD and IHD with the Cys34 disulfide of glutathione and two Cys34 sulfoxidations, were consistent with previous results from smoking and nonsmoking volunteers and nonsmoking women exposed to indoor combustion of coal and wood.

A Preliminary Experimental Examination of Worldview Verification, Perceived Racism, and Stress Reactivity in African Americans

PubMed Central

Lucas, Todd; Lumley, Mark A.; Flack, John M.; Wegner, Rhiana; Pierce, Jennifer; Goetz, Stefan

2016-01-01

Objective According to worldview verification theory, inconsistencies between lived experiences and worldviews are psychologically threatening. These inconsistencies may be key determinants of stress processes that influence cardiovascular health disparities. This preliminary examination considers how experiencing injustice can affect perceived racism and biological stress reactivity among African Americans. Guided by worldview verification theory, it was hypothesized that responses to receiving an unfair outcome would be moderated by fairness of the accompanying decision process, and that this effect would further depend on the consistency of the decision process with preexisting justice beliefs. Method A sample of 118 healthy African American adults completed baseline measures of justice beliefs, followed by a laboratory-based social-evaluative stressor task. Two randomized fairness manipulations were implemented during the task: participants were given either high or low levels of distributive (outcome) and procedural (decision process) justice. Glucocorticoid (cortisol) and inflammatory (C-reactive protein) biological responses were measured in oral fluids, and attributions of racism were also measured. Results The hypothesized 3-way interaction was generally obtained. Among African Americans with a strong belief in justice, perceived racism, cortisol and C-reactive protein responses to low distributive justice were higher when procedural justice was low. Among African Americans with a weak belief in justice however, these responses were higher when a low level of distributive justice was coupled with high procedural justice. Conclusions Biological and psychological processes that contribute to cardiovascular health disparities are affected by consistency between individual-level and contextual justice factors. PMID:27018728

A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina

DOE PAGES

Linger, Jeffrey G.; Taylor, II, Larry E.; Baker, John O.; ...

2015-03-18

One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated amore » native constitutive enolase promoter with the native cbh1 signal sequence. The results are the following: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose demonstrated significantly higher variability in activity. In conclusion, he elimination of background cellulase induction provides much more consistent measured specific activity compared to a traditional cbh1 promoter system induced with lactose. This expression system provides a powerful tool for the expression and comparison of mutant and/or phylogenetically diverse cellobiohydrolases in the industrially relevant cellulase production host H. jecorina.« less

Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

PubMed

Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

2015-04-01

Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm. © 2014 John Wiley & Sons Ltd.

Regulation of the orexigenic neuropeptide, enkephalin, by PPARδ and fatty acids in neurons of the hypothalamus and forebrain.

PubMed

Poon, Kinning; Alam, Mohammad; Karatayev, Olga; Barson, Jessica R; Leibowitz, Sarah F

2015-12-01

Ingestion of a high-fat diet composed mainly of the saturated fatty acid, palmitic (PA), and the unsaturated fatty acid, oleic (OA), stimulates transcription in the brain of the opioid neuropeptide, enkephalin (ENK), which promotes intake of substances of abuse. To understand possible underlying mechanisms, this study examined the nuclear receptors, peroxisome proliferator-activated receptors (PPARs), and tested in hypothalamic and forebrain neurons from rat embryos whether PPARs regulate endogenous ENK and the fatty acids themselves affect these PPARs and ENK. The first set of experiments demonstrated that knocking down PPARδ, but not PPARα or PPARγ, increased ENK transcription, activation of PPARδ by an agonist decreased ENK levels, and PPARδ neurons coexpressed ENK, suggesting that PPARδ negatively regulates ENK. In the second set of experiments, PA treatment of hypothalamic and forebrain neurons had no effect on PPARδ protein while stimulating ENK mRNA and protein, whereas OA increased both mRNA and protein levels of PPARδ in forebrain neurons while having no effect on ENK mRNA and increasing ENK levels. These findings show that PA has a strong, stimulatory effect on ENK and weak effect on PPARδ protein, whereas OA has a strong stimulatory effect on PPARδ and weak effect on ENK, consistent with the inhibitory effect of PPARδ on ENK. They suggest a function for PPARδ, perhaps protective in nature, in embryonic neurons exposed to fatty acids from a fat-rich diet and provide evidence for a mechanism contributing to differential effects of saturated and monounsaturated fatty acids on neurochemical systems involved in consummatory behavior. Our findings show that PPARδ in forebrain and hypothalamic neurons negatively regulates enkephalin (ENK), a peptide known to promote ingestive behavior. This inverse relationship is consistent with our additional findings, that a saturated (palmitic; PA) compared to a monounsaturated fatty acid (oleic; OA) has a strong stimulatory effect on ENK and weak effect on PPARδ. These results suggest that PPARδ protects against the neuronal effects of fatty acids, which differentially affect neurochemical systems involved in ingestive behavior. © 2015 International Society for Neurochemistry.

Functional Analysis of SPINDLY in Gibberellin Signaling in Arabidopsis1[C][W][OA

PubMed Central

Silverstone, Aron L.; Tseng, Tong-Seung; Swain, Stephen M.; Dill, Alyssa; Jeong, Sun Yong; Olszewski, Neil E.; Sun, Tai-ping

2007-01-01

The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-Δ17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-Δ17 phenotype but does not reduce rga-Δ17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification. PMID:17142481

The application of CRISPR technology to high content screening in primary neurons.

PubMed

Callif, Ben L; Maunze, Brian; Krueger, Nick L; Simpson, Matthew T; Blackmore, Murray G

2017-04-01

Axon growth is coordinated by multiple interacting proteins that remain incompletely characterized. High content screening (HCS), in which manipulation of candidate genes is combined with rapid image analysis of phenotypic effects, has emerged as a powerful technique to identify key regulators of axon outgrowth. Here we explore the utility of a genome editing approach referred to as CRISPR (Clustered Regularly Interspersed Palindromic Repeats) for knockout screening in primary neurons. In the CRISPR approach a DNA-cleaving Cas enzyme is guided to genomic target sequences by user-created guide RNA (sgRNA), where it initiates a double-stranded break that ultimately results in frameshift mutation and loss of protein production. Using electroporation of plasmid DNA that co-expresses Cas9 enzyme and sgRNA, we first verified the ability of CRISPR targeting to achieve protein-level knockdown in cultured postnatal cortical neurons. Targeted proteins included NeuN (RbFox3) and PTEN, a well-studied regulator of axon growth. Effective knockdown lagged at least four days behind transfection, but targeted proteins were eventually undetectable by immunohistochemistry in >80% of transfected cells. Consistent with this, anti-PTEN sgRNA produced no changes in neurite outgrowth when assessed three days post-transfection. When week-long cultures were replated, however, PTEN knockdown consistently increased neurite lengths. These CRISPR-mediated PTEN effects were achieved using multi-well transfection and automated phenotypic analysis, indicating the suitability of PTEN as a positive control for future CRISPR-based screening efforts. Combined, these data establish an example of CRISPR-mediated protein knockdown in primary cortical neurons and its compatibility with HCS workflows. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential regulation of ATP binding cassette protein A1 expression and ApoA-I lipidation by Niemann-Pick type C1 in murine hepatocytes and macrophages.

PubMed

Wang, Ming-Dong; Franklin, Vivian; Sundaram, Meenakshi; Kiss, Robert S; Ho, Kenneth; Gallant, Michel; Marcel, Yves L

2007-08-03

Niemann-Pick type C1 (Npc1) protein inactivation results in lipid accumulation in late endosomes and lysosomes, leading to a defect of ATP binding cassette protein A1 (Abca1)-mediated lipid efflux to apolipoprotein A-I (apoA-I) in macrophages and fibroblasts. However, the role of Npc1 in Abca1-mediated lipid efflux to apoA-I in hepatocytes, the major cells contributing to HDL formation, is still unknown. Here we show that, whereas lipid efflux to apoA-I in Npc1-null macrophages is impaired, the lipidation of endogenously synthesized apoA-I by low density lipoprotein-derived cholesterol or de novo synthesized cholesterol or phospholipids in Npc1-null hepatocytes is significantly increased by about 1-, 3-, and 8-fold, respectively. The increased cholesterol efflux reflects a major increase of Abca1 protein in Npc1-null hepatocytes, which contrasts with the decrease observed in Npc1-null macrophages. The increased Abca1 expression is largely post-transcriptional, because Abca1 mRNA is only slightly increased and Lxr alpha mRNA is not changed, and Lxr alpha target genes are reduced. This differs from the regulation of Abcg1 expression, which is up-regulated at both mRNA and protein levels in Npc1-null cells. Abca1 protein translation rate is higher in Npc1-null hepatocytes, compared with wild type hepatocytes as measured by [(35)S]methionine incorporation, whereas there is no difference for the degradation of newly synthesized Abca1 in these two types of hepatocytes. Cathepsin D, which we recently identified as a positive modulator of Abca1, is markedly increased at both mRNA and protein levels by Npc1 inactivation in hepatocytes but not in macrophages. Consistent with this, inhibition of cathepsin D with pepstatin A reduced the Abca1 protein level in both Npc1-inactivated and WT hepatocytes. Therefore, Abca1 expression is specifically regulated in hepatocytes, where Npc1 activity modulates cathepsin D expression and Abca1 protein translation rate.

Single-Molecule Spectroscopy and Imaging Studies of Protein Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter

2012-04-01

Enzymatic reactions and protein-protein interactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. Subtle conformational changes play a crucial role in protein functions, and these protein conformations are highly dynamic rather than being static. We applied AFM-enhanced single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of T4 lysozyme and HPPK enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation. Our new approach is applicable to a wide range of single-molecule FRET measurements for protein conformational changes under enzymatic reactions.

Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat labile enterotoxin in rice endosperm.

PubMed

Oszvald, Maria; Kang, Tae-Jin; Tomoskozi, Sandor; Tamas, Cecilia; Tamas, Laszlo; Kim, Tae-Geum; Yang, Moon-Sik

2007-03-01

Epitopes often require co-delivery with adjuvant and targeting proteins to enable recognition by the immune system, and this approach may also increase the efficacy of the antigen. In this study, we assess and describe the ability of transgenic rice plants to express a fusion protein consisting of the B-subunit of the Escherichia coli heat-labile enterotoxin (LTB) and a synthetic core-neutralizing epitope (COE) of porcine epidemic diarrhea virus (PEDV), inducing an enteric disease that is seen most predominantly in piglets. Both components of the fusion proteins were detected with Western blot analysis. The fusion protein was determined to assemble into pentamers, as was evidenced by its ability to bind to GM1 gangliosides, and evidenced an average level of expression in a transgenic rice endosperm. This indicates that the expression system of the plant is capable of generating a sizable amount of antigen, possibly allowing for the successful development of an edible vaccine.

Tertiary structural propensities reveal fundamental sequence/structure relationships.

PubMed

Zheng, Fan; Zhang, Jian; Grigoryan, Gevorg

2015-05-05

Extracting useful generalizations from the continually growing Protein Data Bank (PDB) is of central importance. We hypothesize that the PDB contains valuable quantitative information on the level of local tertiary structural motifs (TERMs). We show that by breaking a protein structure into its constituent TERMs, and querying the PDB to characterize the natural ensemble matching each, we can estimate the compatibility of the structure with a given amino acid sequence through a metric we term "structure score." Considering submissions from recent Critical Assessment of Structure Prediction (CASP) experiments, we found a strong correlation (R = 0.69) between structure score and model accuracy, with poorly predicted regions readily identifiable. This performance exceeds that of leading atomistic statistical energy functions. Furthermore, TERM-based analysis of two prototypical multi-state proteins rapidly produced structural insights fully consistent with prior extensive experimental studies. We thus find that TERM-based analysis should have considerable utility for protein structural biology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Salivary sIg-A response against the recombinant Ag38 antigen of Mycobacterium tuberculosis Indonesian strain.

PubMed

Raras, Tri Yudani Mardining; Sholeh, Gamal; Lyrawati, Diana

2014-01-01

An evaluation of the humoral response based on secretory immunoglobulin A levels in the saliva of pulmonary tuberculosis (TB) acid-fast bacillus-positive (TB-AFB+) patients against a recombinant 38 kDa antigen (Ag38-rec) is reported. A total of 60 saliva samples consist of 30 TB-AFB+ patients and 30 healthy controls were tested against 500 ng of semi-purified antigen using the dot blot method. Results showed that the protein antigen could differentiate between healthy individuals and TB-AFB(+) patients. Whole saliva demonstrated better reactivity than centrifuged saliva. The Ag38-rec protein indicated statistically comparable sensitivity (80% versus 90%), but lower specificity (36.6% versus 70%) compared with purified protein derivative (PPD). Surprisingly, both antigens similarly recognized secretory immunoglobulin A in the saliva of the healthy group (50% versus 50%, respectively). These findings suggest that the Ag38-rec protein originating from a local strain of Mycobacterium tuberculosis may be used for TB screening, however require purity improvement.

Expression-Linked Patterns of Codon Usage, Amino Acid Frequency, and Protein Length in the Basally Branching Arthropod Parasteatoda tepidariorum

PubMed Central

Whittle, Carrie A.; Extavour, Cassandra G.

2016-01-01

Abstract Spiders belong to the Chelicerata, the most basally branching arthropod subphylum. The common house spider, Parasteatoda tepidariorum, is an emerging model and provides a valuable system to address key questions in molecular evolution in an arthropod system that is distinct from traditionally studied insects. Here, we provide evidence suggesting that codon usage, amino acid frequency, and protein lengths are each influenced by expression-mediated selection in P. tepidariorum. First, highly expressed genes exhibited preferential usage of T3 codons in this spider, suggestive of selection. Second, genes with elevated transcription favored amino acids with low or intermediate size/complexity (S/C) scores (glycine and alanine) and disfavored those with large S/C scores (such as cysteine), consistent with the minimization of biosynthesis costs of abundant proteins. Third, we observed a negative correlation between expression level and coding sequence length. Together, we conclude that protein-coding genes exhibit signals of expression-related selection in this emerging, noninsect, arthropod model. PMID:27017527

In vivo binding of hot pepper bZIP transcription factor CabZIP1 to the G-box region of pathogenesis-related protein 1 promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Boo-Ja; Park, Chang-Jin; Kim, Sung-Kyu

2006-05-26

We find that salicylic acid and ethephon treatment in hot pepper increases the expression of a putative basic/leucine zipper (bZIP) transcription factor gene, CabZIP1. CabZIP1 mRNA is expressed ubiquitously in various organs. The green fluorescent protein-fused transcription factor, CabZIP1::GFP, can be specifically localized to the nucleus, an action that is consistent with the presence of a nuclear localization signal in its protein sequence. Transient overexpression of the CabZIP1 transcription factor results in an increase in PR-1 transcripts level in Nicotiana benthamiana leaves. Using chromatin immunoprecipitation, we demonstrate that CabZIP1 binds to the G-box elements in native promoter of the hotmore » pepper pathogenesis-related protein 1 (CaPR-1) gene in vivo. Taken together, our results suggest that CabZIP1 plays a role as a transcriptional regulator of the CaPR-1 gene.« less

FliH and FliI ensure efficient energy coupling of flagellar type III protein export in Salmonella.

PubMed

Minamino, Tohru; Kinoshita, Miki; Inoue, Yumi; Morimoto, Yusuke V; Ihara, Kunio; Koya, Satomi; Hara, Noritaka; Nishioka, Noriko; Kojima, Seiji; Homma, Michio; Namba, Keiichi

2016-06-01

For construction of the bacterial flagellum, flagellar proteins are exported via its specific export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane (TM) export gate complex and a cytoplasmic ATPase complex consisting of FliH, FliI, and FliJ. FlhA is a TM export gate protein and plays important roles in energy coupling of protein translocation. However, the energy coupling mechanism remains unknown. Here, we performed a cross-complementation assay to measure robustness of the energy transduction system of the export apparatus against genetic perturbations. Vibrio FlhA restored motility of a Salmonella ΔflhA mutant but not that of a ΔfliH-fliI flhB(P28T) ΔflhA mutant. The flgM mutations significantly increased flagellar gene expression levels, allowing Vibrio FlhA to exert its export activity in the ΔfliH-fliI flhB(P28T) ΔflhA mutant. Pull-down assays revealed that the binding affinities of Vibrio FlhA for FliJ and the FlgN-FlgK chaperone-substrate complex were much lower than those of Salmonella FlhA. These suggest that Vibrio FlhA requires the support of FliH and FliI to efficiently and properly interact with FliJ and the FlgN-FlgK complex. We propose that FliH and FliI ensure robust and efficient energy coupling of protein export during flagellar assembly. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Delineating slowly and rapidly evolving fractions of the Drosophila genome.

PubMed

Keith, Jonathan M; Adams, Peter; Stephen, Stuart; Mattick, John S

2008-05-01

Evolutionary conservation is an important indicator of function and a major component of bioinformatic methods to identify non-protein-coding genes. We present a new Bayesian method for segmenting pairwise alignments of eukaryotic genomes while simultaneously classifying segments into slowly and rapidly evolving fractions. We also describe an information criterion similar to the Akaike Information Criterion (AIC) for determining the number of classes. Working with pairwise alignments enables detection of differences in conservation patterns among closely related species. We analyzed three whole-genome and three partial-genome pairwise alignments among eight Drosophila species. Three distinct classes of conservation level were detected. Sequences comprising the most slowly evolving component were consistent across a range of species pairs, and constituted approximately 62-66% of the D. melanogaster genome. Almost all (>90%) of the aligned protein-coding sequence is in this fraction, suggesting much of it (comprising the majority of the Drosophila genome, including approximately 56% of non-protein-coding sequences) is functional. The size and content of the most rapidly evolving component was species dependent, and varied from 1.6% to 4.8%. This fraction is also enriched for protein-coding sequence (while containing significant amounts of non-protein-coding sequence), suggesting it is under positive selection. We also classified segments according to conservation and GC content simultaneously. This analysis identified numerous sub-classes of those identified on the basis of conservation alone, but was nevertheless consistent with that classification. Software, data, and results available at www.maths.qut.edu.au/-keithj/. Genomic segments comprising the conservation classes available in BED format.

Visualizing and Clustering Protein Similarity Networks: Sequences, Structures, and Functions.

PubMed

Mai, Te-Lun; Hu, Geng-Ming; Chen, Chi-Ming

2016-07-01

Research in the recent decade has demonstrated the usefulness of protein network knowledge in furthering the study of molecular evolution of proteins, understanding the robustness of cells to perturbation, and annotating new protein functions. In this study, we aimed to provide a general clustering approach to visualize the sequence-structure-function relationship of protein networks, and investigate possible causes for inconsistency in the protein classifications based on sequences, structures, and functions. Such visualization of protein networks could facilitate our understanding of the overall relationship among proteins and help researchers comprehend various protein databases. As a demonstration, we clustered 1437 enzymes by their sequences and structures using the minimum span clustering (MSC) method. The general structure of this protein network was delineated at two clustering resolutions, and the second level MSC clustering was found to be highly similar to existing enzyme classifications. The clustering of these enzymes based on sequence, structure, and function information is consistent with each other. For proteases, the Jaccard's similarity coefficient is 0.86 between sequence and function classifications, 0.82 between sequence and structure classifications, and 0.78 between structure and function classifications. From our clustering results, we discussed possible examples of divergent evolution and convergent evolution of enzymes. Our clustering approach provides a panoramic view of the sequence-structure-function network of proteins, helps visualize the relation between related proteins intuitively, and is useful in predicting the structure and function of newly determined protein sequences.

Fast and Accurate Protein False Discovery Rates on Large-Scale Proteomics Data Sets with Percolator 3.0.

PubMed

The, Matthew; MacCoss, Michael J; Noble, William S; Käll, Lukas

2016-11-01

Percolator is a widely used software tool that increases yield in shotgun proteomics experiments and assigns reliable statistical confidence measures, such as q values and posterior error probabilities, to peptides and peptide-spectrum matches (PSMs) from such experiments. Percolator's processing speed has been sufficient for typical data sets consisting of hundreds of thousands of PSMs. With our new scalable approach, we can now also analyze millions of PSMs in a matter of minutes on a commodity computer. Furthermore, with the increasing awareness for the need for reliable statistics on the protein level, we compared several easy-to-understand protein inference methods and implemented the best-performing method-grouping proteins by their corresponding sets of theoretical peptides and then considering only the best-scoring peptide for each protein-in the Percolator package. We used Percolator 3.0 to analyze the data from a recent study of the draft human proteome containing 25 million spectra (PM:24870542). The source code and Ubuntu, Windows, MacOS, and Fedora binary packages are available from http://percolator.ms/ under an Apache 2.0 license. Graphical Abstract ᅟ.

Activity of calcium activated protease in skeletal muscles and its changes in atrophy and stretch

NASA Technical Reports Server (NTRS)

Ellis, S.; Nagainis, P. A.

1984-01-01

The reduction of protein content in skeletal muscle undergoing disuse-induced atrophy is correlated with accelerated rates of protein degradation and reduced rates of protein synthesis (Goldspink, 1977). It is not known in what manner myofibers are partially disassembled during disuse atrophy to fibers of smaller diameter; nor is it known which proteases are responsible for this morphological change in contractile protein mass. Dayton and colleagues (1975) have suggested that the Ca(2+)-activated protease (CaP) may initiate myofibril degradation. The discovery of a form of CaP that is activatable by nano-molar concentrations of Ca(2+) indicates that CaP activity may be regulated by physiological concentrations of Ca(2+) (Mellgren, 1980). The enhancement of proteolysis by the Ca(2+) ionophore A23187, reported by Etlinger (1979), is consistent with a significant role for CaP in protein degradation. It was of interest, therefore, to measure the levels of CaP activity and the CaP inhibitor in extracts obtained from skeletal muscles of rat and chicken limbs undergoing disuse atrophy or stretch hypertrophy, respectively.

Rehabilitation in severe memory deficit: A case study

PubMed Central

Sousa, Nariana Mattos Figueiredo

2017-01-01

The term amnesia refers to a pathological state of mind in which memory and learning are affected to a greater extent than other cognitive functions in a patient without altered level of consciousness. The aim of the current study was to describe a case of severe amnesia in a patient during neurological rehabilitation and to report the importance of preserved cognitive functions to compensate for the mnemonic deficit. VJA presented a clinical condition suggestive of encephalopathy due to caloric-protein malnutrition following several abdominal surgical procedures for complicated choledocholithiasis. A descriptive analysis of the results was carried out to outline the goals attained and the factors limiting implementation of memory aids. After the intervention program, consisting of individual and group activities, VJA showed improvement in level of recall with repetition of tasks, but still required constant external monitoring. Longitudinal follow-up is necessary to obtain more consistent results. PMID:29213515

Rehabilitation in severe memory deficit: A case study.

PubMed

Sousa, Nariana Mattos Figueiredo

2017-01-01

The term amnesia refers to a pathological state of mind in which memory and learning are affected to a greater extent than other cognitive functions in a patient without altered level of consciousness. The aim of the current study was to describe a case of severe amnesia in a patient during neurological rehabilitation and to report the importance of preserved cognitive functions to compensate for the mnemonic deficit. VJA presented a clinical condition suggestive of encephalopathy due to caloric-protein malnutrition following several abdominal surgical procedures for complicated choledocholithiasis. A descriptive analysis of the results was carried out to outline the goals attained and the factors limiting implementation of memory aids. After the intervention program, consisting of individual and group activities, VJA showed improvement in level of recall with repetition of tasks, but still required constant external monitoring. Longitudinal follow-up is necessary to obtain more consistent results.

Influence of essential oil of Hyssopus officinalis on the chemical composition of the walls of Aspergillus fumigatus (Fresenius).

PubMed

Ghfir, B; Fonvieille, J L; Dargent, R

1997-07-01

The cell walls of the growing hyphae of Aspergillus fumigatus (Fresenius) cultured in the presence or absence of the essential oil of Hyssopus officinalis were isolated and their chemical composition analysed. The presence of the essential oil led to a reduction in levels of neutral sugars, uronic acid and proteins, whereas amino sugars, lipids and phosphorus levels were increased. HPLC analysis of the neutral sugars showed that they consisted mainly of glucose, mannose and galactose, while the amino sugars consisted of glucosamine and galactosamine. The presence of the essential oil in the culture medium induced marked changes in the content of galactose and galactosamine. Cell walls were fractionated by treatment with alkali and acid. The essential oil induced similar alterations in the various fractions with a more marked effect on the major constituents. The alterations were related to changes in the structure of the cells.

Measurement of lipocortin 1 levels in murine peripheral blood leukocytes by flow cytometry: modulation by glucocorticoids and inflammation.

PubMed

Perretti, M; Flower, R J

1996-06-01

1. Lipocortin 1 (LC1) immunoreactivity in murine peripheral blood leukocytes was quantified by use of a flow cytometric technique associated with a permeabilisation protocol with saponin. Using specific antisera raised against the whole protein or against its N-terminus peptide, cell-associated LC1-like immunoreactivity was easily detected in circulating neutrophils and monocytes, whereas very low levels were found in lymphocytes. Of the total protein measured 17.6% and 36% were associated with the external plasma membrane in neutrophils and monocytes, as assessed in the absence of cell permeabilisation, whereas no signal was detected on lymphocyte plasma membrane. 2. Treatment of mice with dexamethasone (Dex; 0.5-5 micrograms per mouse corresponding to approximately 0.015-1.5 mg kg-1) increased LC1 levels in neutrophils and monocytes. The 2-3 fold increase in LC1 levels was time-dependent with a peak at 2 h. Treatment of mice with the steroid antagonist, RU486 (two doses of 20 mg kg-1 orally) decreased LC1-like immunoreactivity in all three types of circulating leukocytes by > or = 50%. 3. Extravasation of blood neutrophils into inflamed tissue sites resulted in a consistent reduction (> or = 50%) in LC1 levels compared with circulating neutrophils. A high LC1-like immunoreactivity was also measured in resident macrophages, of which approximately one third was membrane-associated. Induction of an acute inflammatory response in the murine peritoneal cavity did not modify total LC1 levels measured in macrophages, but reduced membrane-associated LC1 to a significant extent, i.e. up to 70%. 4. In conclusion, flow cytometric analysis is a rapid and convenient method for detecting and measuring LC1 in murine leukocytes. We confirmed that LC1 protein expression is controlled by exogenous and endogenous glucocorticoids. Amongst other factor(s) influencing protein concentrations, extravasation was found to be associated with a reduced LC1 expression in the emigrated cells.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Feng; Zhang, Yuanyuan; Wang, Qingzhu

Objective: This study was designed to investigate the protective effect of forkhead transcription factor O1 (FoxO1) on podocyte injury in rats with diabetic nephropathy. Methods: Streptozotocin-induced diabetic rats were served as DM group, while DM rats transfected with blank lentiviral vectors (LV-pSC-GFP) or lentiviral vectors carrying constitutively active FoxO1 (LV-CA-FoxO1) were served as LV-NC group or LV-CA group, respectively. The control group (NG) consisted of uninduced rats that received an injection of diluent buffer. At 2, 4, and 8 weeks after transfection, the levels of urine albumin, blood glucose, blood urea nitrogen, serum creatinine and urine podocalyxin were measured. Real-timemore » PCR and western blotting were performed to measure mRNA and protein levels of FoxO1, podocalyxin, nephrin, and desmin in renal cortex. In addition, light and electron microscopy were used to detect structural changes in the glomerulus and podocytes. Results: Compared with the rats in LV-NC and DM groups, LV-CA rats showed a significant increase in FoxO1 mRNA and protein levels and a distinct decrease in urine albumin, blood urea nitrogen, and serum creatinine (except at the two-week time point) levels (p < 0.05). Podocalyxin and nephrin mRNA and protein levels increased (p < 0.05), whereas desmin mRNA and protein levels decreased (p < 0.05). Pathological changes in glomerulus were also ameliorated in LV-CA group. Conclusions: Upregulating expression of FoxO1 by transduction with recombinant lentivirus ameliorates podocyte injury in diabetic rats. - Highlights: • The structures and functions of podocytes were impaired in STZ-induced diabetic rats. • Constitutively active FoxO1 ameliorates structure injury and preserves function of podocytes in diabetic rats. • FoxO1 may alleviate the pathological changes associated with diabetic nephropathy.« less

Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein

PubMed Central

2004-01-01

The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30–90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA. PMID:15496143

Reactive Oxygen Species Alter Autocrine and Paracrine Signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zangar, Richard C.; Bollinger, Nikki; Weber, Thomas J.

2011-12-01

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endo-plasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a varietymore » of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A customELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-{kappa}B pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.« less

Topology-based modeling of intrinsically disordered proteins: balancing intrinsic folding and intermolecular interactions.

PubMed

Ganguly, Debabani; Chen, Jianhan

2011-04-01

Coupled binding and folding is frequently involved in specific recognition of so-called intrinsically disordered proteins (IDPs), a newly recognized class of proteins that rely on a lack of stable tertiary fold for function. Here, we exploit topology-based Gō-like modeling as an effective tool for the mechanism of IDP recognition within the theoretical framework of minimally frustrated energy landscape. Importantly, substantial differences exist between IDPs and globular proteins in both amino acid sequence and binding interface characteristics. We demonstrate that established Gō-like models designed for folded proteins tend to over-estimate the level of residual structures in unbound IDPs, whereas under-estimating the strength of intermolecular interactions. Such systematic biases have important consequences in the predicted mechanism of interaction. A strategy is proposed to recalibrate topology-derived models to balance intrinsic folding propensities and intermolecular interactions, based on experimental knowledge of the overall residual structure level and binding affinity. Applied to pKID/KIX, the calibrated Gō-like model predicts a dominant multistep sequential pathway for binding-induced folding of pKID that is initiated by KIX binding via the C-terminus in disordered conformations, followed by binding and folding of the rest of C-terminal helix and finally the N-terminal helix. This novel mechanism is consistent with key observations derived from a recent NMR titration and relaxation dispersion study and provides a molecular-level interpretation of kinetic rates derived from dispersion curve analysis. These case studies provide important insight into the applicability and potential pitfalls of topology-based modeling for studying IDP folding and interaction in general. Copyright © 2011 Wiley-Liss, Inc.

Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Bin, E-mail: binli@unmc.edu; Eyer, Peter, E-mail: peter.eyer@lrz.uni-muenchen.de; Eddleston, Michael, E-mail: M.Eddleston@ed.ac.uk

2013-06-15

Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasmamore » levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.« less

Texturized dairy proteins.

PubMed

Onwulata, Charles I; Phillips, John G; Tunick, Michael H; Qi, Phoebi X; Cooke, Peter H

2010-03-01

Dairy proteins are amenable to structural modifications induced by high temperature, shear, and moisture; in particular, whey proteins can change conformation to new unfolded states. The change in protein state is a basis for creating new foods. The dairy products, nonfat dried milk (NDM), whey protein concentrate (WPC), and whey protein isolate (WPI) were modified using a twin-screw extruder at melt temperatures of 50, 75, and 100 degrees C, and moistures ranging from 20 to 70 wt%. Viscoelasticity and solubility measurements showed that extrusion temperature was a more significant (P < 0.05) change factor than moisture content. The degree of texturization, or change in protein state, was characterized by solubility (R(2)= 0.98). The consistency of the extruded dairy protein ranged from rigid (2500 N) to soft (2.7 N). Extruding at or above 75 degrees C resulted in increased peak force for WPC (138 to 2500 N) and WPI (2.7 to 147.1 N). NDM was marginally texturized; the presence of lactose interfered with its texturization. WPI products extruded at 50 degrees C were not texturized; their solubility values ranged from 71.8% to 92.6%. A wide possibility exists for creating new foods with texturized dairy proteins due to the extensive range of states achievable. Dairy proteins can be used to boost the protein content in puffed snacks made from corn meal, but unmodified, they bind water and form doughy pastes with starch. To minimize the water binding property of dairy proteins, WPI, or WPC, or NDM were modified by extrusion processing. Extrusion temperature conditions were adjusted to 50, 75, or 100 degrees C, sufficient to change the structure of the dairy proteins, but not destroy them. Extrusion modified the structures of these dairy proteins for ease of use in starchy foods to boost nutrient levels. Dairy proteins can be used to boost the protein content in puffed snacks made from corn meal, but unmodified, they bind water and form doughy pastes with starch. To minimize the water binding property of dairy proteins, whey protein isolate, whey protein concentrate, or nonfat dried milk were modified by extrusion processing. Extrusion temperature conditions were adjusted to 50, 75, or 100 degrees C, sufficient to change the structure of the dairy proteins, but not destroy them. Extrusion modified the structures of these dairy proteins for ease of use in starchy foods to boost nutrient levels.

Effects of nutrition level of concentrate-based diets on growth performance and carcass characteristics of Hainan black goats.

PubMed

Wang, Dingfa; Zhou, Luli; Zhou, Hanlin; Hou, Guanyu; Li, Mao; Shi, Liguang; Huang, Xianzhou; Guan, Song

2014-06-01

This study assessed the effects of different nutrition levels of diets on growth performance and carcass characteristics of Hainan black goat. Twenty-four goats were divided into six diet treatments, which consisted of two levels of crude protein (CP; 15 and 17 %) and three levels of digestive energy (DE; 11.72, 12.55, and 13.39 MJ/kg). The results revealed that 17 % CP significantly (P < 0.05) increased ADG and improved FCR compared with 15 % CP. Therefore, the CP levels of diet affected growth performance. CP and DE levels in the diet had no significant effects (P > 0.05) on carcass characteristics of the goats. The mRNA expression levels of insulin-like growth factor 1 in muscle tissues increased with increasing CP and DE levels (P < 0.05).

Changes in PEP carboxylase, rubisco and rubisco activase mRNA levels from maize (Zea mays) exposed to a chronic ozone stress.

PubMed

Leitao, Louis; Maoret, Jean-José; Biolley, Jean-Philippe

2007-01-01

We quantified the ozone impact on levels of Zea mays L. cv. Chambord mRNAs encoding C4-phosphoenolpyruvate carboxylase (C4-PEPc), ribulose-l,5-bisphosphate carboxylase/oxygenase small and large subunits (Rubisco-SSU and Rubisco-LSU, respectively) and Rubisco activase (RCA) using real-time RT-PCR. Foliar pigment content, PEPc and Rubisco protein amounts were simultaneously determined. Two experiments were performed to study the ozone response of the 5th and the 10th leaf. For each experiment, three ozone concentrations were tested in open-top chambers: non-filtered air (NF, control) and non-filtered air containing 40 (+40) and 80 nL L-1 (+80) ozone. Regarding the 5th leaf, +40 atmosphere induced a loss in pigmentation, PEPc and Rubisco activase mRNAs. However, it was unable to notably depress carboxylase protein amounts and mRNAs encoding Rubisco. Except for Rubisco mRNAs, all other measured parameters from 5th leaf were depressed by +80 atmosphere. Regarding the 10th leaf, +40 atmosphere increased photosynthetic pigments and transcripts encoding Rubisco and Rubisco activase. Rubisco and PEPc protein amounts were not drastically changed, even if they tended to be increased. Level of C4-PEPc mRNA remained almost stable. In response to +80 atmosphere, pigments and transcripts encoding PEPc were notably decreased. Rubisco and PEPc protein amounts also declined to a lesser extent. Conversely, the level of transcripts encoding both Rubisco subunits and Rubisco activase that were not consistently disturbed tended to be slightly augmented. So, the present study suggests that maize leaves can respond differentially to a similar ozone stress.

HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444 issue 3)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiaozhen; Institute of Systems Biology, Peking University, Beijing 100191; Lu, Guang

2014-02-14

Highlights: • The 2000–2634 aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the controlmore » of BRCA1 protein level. HUWE1binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.« less

HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444, isse 4)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiaozhen; Institute of Systems Biology, Peking University, Beijing 100191; Lu, Guang

2014-02-21

Highlights: • The 2000–2634aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control ofmore » BRCA1 protein level. HUWE1 binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.« less

Recognition of the different structural forms of the capsid protein determines the outcome following infection with porcine circovirus type 2.

PubMed

Trible, Benjamin R; Suddith, Andrew W; Kerrigan, Maureen A; Cino-Ozuna, Ada G; Hesse, Richard A; Rowland, Raymond R R

2012-12-01

Porcine circovirus type 2 (PCV2) capsid protein (CP) is the only protein necessary for the formation of the virion capsid, and recombinant CP spontaneously forms virus-like particles (VLPs). Located within a single CP subunit is an immunodominant epitope consisting of residues 169 to 180 [CP(169-180)], which is exposed on the surface of the subunit, but, in the structural context of the VLP, the epitope is buried and inaccessible to antibody. High levels of anti-CP(169-180) activity are associated with porcine circovirus-associated disease (PCVAD). The purpose of this study was to investigate the role of the immune response to monomer CP in the development of PCVAD. The approach was to immunize pigs with CP monomer, followed by challenge with PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV). To maintain the CP immunogen as a stable monomer, CP(43-233) was fused to ubiquitin (Ub-CP). Size exclusion chromatography showed that Ub-CP was present as a single 33-kDa protein. Pigs immunized with Ub-CP developed a strong antibody response to PCV2, including antibodies against CP(169-180). However, only low levels of virus neutralizing activity were detected, and viremia levels were similar to those of nonimmunized pigs. As a positive control, immunization with baculovirus-expressed CP (Bac-CP) resulted in high levels of virus neutralizing activity, small amounts of anti-CP(169-180) activity, and the absence of viremia in pigs following virus challenge. The data support the role of CP(169-180) as an immunological decoy and illustrate the importance of the structural form of the CP immunogen in determining the outcome following infection.

Effect of statins on sirtuin 1 and endothelial nitric oxide synthase expression in young patients with a history of premature myocardial infarction.

PubMed

Yamaç, Aylin Hatice; Kılıç, Ülkan

2018-04-01

The present study was an investigation of the effect of statins on the expression of circulating sirtuin 1 (SIRT1) and endothelial nitric oxide synthase (eNOS) proteins, and on the distribution of single nucleotide polymorphisms (SNPs) of the SIRT1 gene in patients with a history of premature myocardial infarction (PMI). A total of 108 patients who had suffered from a premature ST-elevation myocardial infarction (STEMI) under the age of 45 years were enrolled in this study. While 79 patients had been taking statins since the index event, 29 patients had discontinued statin treatment after hospital discharge due to noncompliance or insufficient information about the importance of continuous statin therapy in post-MI patients. The control group consisted of 91 healthy patients without a previous cardiovascular event. The levels of SIRT1 and eNOS protein; oxidative stress markers, like total antioxidant status (TAS), total oxidant status (TOS), and the oxidative stress index (OSI); as well as the distribution of the SNPs rs7069102 and rs2273773 were measured and analyzed. A significant increase in the SIRT1 level (p<0.001) and a significant decrease in the eNOS level (p=0.001) was observed in all genotypes and alleles for both SNPs in patients who received statin therapy compared with the control group. Both SNPs were distributed in a similar frequency in the 2 MI groups, irrespective of statin treatment. Statins induce SIRT1 protein, which might have a cardioprotective role after PMI. In addition, the eNOS protein level was low in all of the MI patients, suggesting that impairment of eNOS expression is disease-specific without a causal link to SIRT1.

A high-protein diet during hospitalization is associated with an accelerated decrease in soluble urokinase plasminogen activator receptor levels in acutely ill elderly medical patients with SIRS.

PubMed

Tavenier, Juliette; Haupt, Thomas H; Andersen, Aino L; Buhl, Sussi F; Langkilde, Anne; Andersen, Jens R; Jensen, Jens-Erik B; Pedersen, Mette M; Petersen, Janne; Andersen, Ove

2017-05-01

Acute illness and hospitalization in elderly individuals are often accompanied by the systemic inflammatory response syndrome (SIRS) and malnutrition, both associated with wasting and mortality. Nutritional support and resistance training were shown to increase muscle anabolism and reduce inflammation in healthy elderly. We hypothesized that nutritional support and resistance training would accelerate the resolution of inflammation in hospitalized elderly patients with SIRS. Acutely admitted patients aged >65 years with SIRS were randomized to an intervention consisting of a high-protein diet (1.7 g/kg per day) during hospitalization, and daily protein supplement (18.8 g) and 3 weekly resistance training sessions for 12 weeks after discharge (Intervention, n=14), or to standard-care (Control, n=15). Plasma levels of the inflammatory biomarkers soluble urokinase plasminogen activator receptor (suPAR), interleukin-6, C-reactive protein (CRP), and albumin were measured at admission, discharge, and 4 and 13 weeks after discharge. The Intervention group had an earlier decrease in suPAR levels than the Control group: -15.4% vs. +14.5%, P=.007 during hospitalization, and -2.4% vs. -28.6%, P=.007 between discharge and 4 weeks. There were no significant effects of the intervention on the other biomarkers. All biomarkers improved significantly between admission and 13 weeks, although with different kinetics (suPAR: -22%, interleukin-6: -86%, CRP: -89%, albumin: +11%). Nutritional support during hospitalization was associated with an accelerated decrease in suPAR levels, whereas the combined nutrition and resistance training intervention after discharge did not appear to affect the inflammatory state. Our results indicate that improved nutritional care during hospitalization may accelerate recovery in acutely ill elderly medical patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Palmitate-induced ER stress and inhibition of protein synthesis in cultured myotubes does not require Toll-like receptor 4.

PubMed

Perry, Ben D; Rahnert, Jill A; Xie, Yang; Zheng, Bin; Woodworth-Hobbs, Myra E; Price, S Russ

2018-01-01

Saturated fatty acids, such as palmitate, are elevated in metabolically dysfunctional conditions like type 2 diabetes mellitus. Palmitate has been shown to impair insulin sensitivity and suppress protein synthesis while upregulating proteolytic systems in skeletal muscle. Increased sarco/endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response may contribute to the palmitate-induced impairment of muscle protein synthesis. In some cell types, ER stress occurs through activation of the Toll-like receptor 4 (TLR4). Given the link between ER stress and suppression of protein synthesis, we investigated whether palmitate induces markers of ER stress and protein synthesis by activating TLR4 in cultured mouse C2C12 myotubes. Myotubes were treated with vehicle, a TLR4-specific ligand (lipopolysaccharides), palmitate, or a combination of palmitate plus a TLR4-specific inhibitor (TAK-242). Inflammatory indicators of TLR4 activation (IL-6 and TNFα) and markers of ER stress were measured, and protein synthesis was assessed using puromycin incorporation. Palmitate substantially increased the levels of IL-6, TNF-α, CHOP, XBP1s, and ATF 4 mRNAs and augmented the levels of CHOP, XBP1s, phospho-PERK and phospho-eIF2α proteins. The TLR4 antagonist attenuated both acute palmitate and LPS-induced increases in IL-6 and TNFα, but did not reduce ER stress signaling with either 6 h or 24 h palmitate treatment. Similarly, treating myotubes with palmitate for 6 h caused a 43% decline in protein synthesis consistent with an increase in phospho-eIF2α, and the TLR4 antagonist did not alter these responses. These results suggest that palmitate does not induce ER stress through TLR4 in muscle, and that palmitate impairs protein synthesis in skeletal muscle in part by induction of ER stress.

Substitution of common concentrates with by-products modulated ruminal fermentation, nutrient degradation, and microbial community composition in vitro.

PubMed

Ertl, P; Knaus, W; Metzler-Zebeli, B U; Klevenhusen, F; Khiaosa-Ard, R; Zebeli, Q

2015-07-01

A rumen simulation technique was used to evaluate the effects of the complete substitution of a common concentrate mixture (CON) with a mixture consisting solely of by-products from the food industry (BP) at 2 different forage-to-concentrate ratios on ruminal fermentation profile, nutrient degradation, and abundance of rumen microbiota. The experiment was a 2×2 factorial arrangement with 2 concentrate types (CON and BP) and 2 concentrate levels (25 and 50% of diet dry matter). The experiment consisted of 2 experimental runs with 12 fermentation vessels each (n=6 per treatment). Each run lasted for 10d, with data collection on the last 5d. The BP diets had lower starch, but higher neutral detergent fiber (NDF) and fat contents compared with CON. Degradation of crude protein was decreased, but NDF and nonfiber carbohydrate degradation were higher for the BP diets. At the 50% concentrate level, organic matter degradation tended to be lower for BP and CH4 formation per unit of NDF degraded was also lower for BP. The BP mixture led to a higher concentration of propionate and a lower acetate-to-propionate ratio, whereas concentrations of butyrate and caproate decreased. Concentrate type did not affect microbial community composition, except that the abundance of bacteria of the genus Prevotella was higher for BP. Increasing the concentrate level resulted in higher degradation of organic matter and crude protein. At the higher concentrate level, total short-chain fatty acid formation increased and concentrations of isobutyrate and valerate decreased. In addition, at the 50% concentrate level, numbers of protozoa increased, whereas numbers of methanogens, anaerobic fungi, and fibrolytic bacteria decreased. No interaction was noted between the 2 dietary factors on most variables, except that at the higher concentrate level the effects of BP on CH4 and CO2 formation per unit of NDF degraded, crude protein degradation, and the abundance of Prevotella were more prominent. In conclusion, the results of this study suggest that BP in the diet can adequately substitute CON with regard to ruminal fermentation profile and microbiota, showing even favorable fermentation patterns when fed at 50% inclusion rate. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Disturbed secretion of mutant adiponectin associated with the metabolic syndrome.

PubMed

Kishida, Ken; Nagaretani, Hiroyuki; Kondo, Hidehiko; Kobayashi, Hideki; Tanaka, Sachiyo; Maeda, Norikazu; Nagasawa, Azumi; Hibuse, Toshiyuki; Ohashi, Koji; Kumada, Masahiro; Nishizawa, Hitoshi; Okamoto, Yoshihisa; Ouchi, Noriyuki; Maeda, Kazuhisa; Kihara, Shinji; Funahashi, Tohru; Matsuzawa, Yuji

2003-06-20

Adiponectin, an adipocyte-derived protein, consists of collagen-like fibrous and complement C1q-like globular domains, and circulates in human plasma in a multimeric form. The protein exhibits anti-diabetic and anti-atherogenic activities. However, adiponectin plasma concentrations are low in obese subjects, and hypoadiponectinemia is associated with the metabolic syndrome, which is a cluster of insulin resistance, type 2 diabetes mellitus, hypertension, and dyslipidemia. We have recently reported a missense mutation in the adiponectin gene, in which isoleucine at position 164 in the globular domain is substituted with threonine (I164T). Subjects with this mutation showed markedly low level of plasma adiponectin and clinical features of the metabolic syndrome. Here, we examined the molecular characteristics of the mutant protein associated with a genetic cause of hypoadiponectinemia. The current study revealed (1) the mutant protein showed an oligomerization state similar to the wild-type as determined by gel filtration chromatography and, (2) the mutant protein exhibited normal insulin-sensitizing activity, but (3) pulse-chase study showed abnormal secretion of the mutant protein from adipose tissues. Our results suggest that I164T mutation is associated with hypoadiponectinemia through disturbed secretion into plasma, which may contribute to the development of the metabolic syndrome.

De novo disruption of promoter and exon 1 of STAR gene reveals essential role for gonadal development.

PubMed

Piya, Anil; Kaur, Jasmeet; Rice, Alan M; Bose, Himangshu S

2017-01-01

Cholesterol transport into the mitochondria is required for synthesis of the first steroid, pregnenolone. Cholesterol is transported by the steroidogenic acute regulatory protein (STAR), which acts at the outer mitochondrial membrane prior to its import. Mutations in the STAR protein result in lipoid congenital adrenal hyperplasia (CAH). Although the STAR protein consists of seven exons, biochemical analysis in nonsteroidogenic COS-1 cells showed that the first two were not essential for pregnenolone synthesis. Here, we present a patient with ambiguous genitalia, salt-lossing crisis within two weeks after birth and low cortisol levels. Sequence analysis of the STAR , including the exon-intron boundaries, showed the complete deletion of exon 1 as well as more than 50 nucleotides upstream of STAR promoter. Mitochondrial protein import with the translated protein through synthesis cassette of the mutant STAR lacking exon 1 showed protein translation, but it is less likely to have synthesized without a promoter in our patient. Thus, a full-length STAR gene is necessary for physiological mitochondrial cholesterol transport in vivo . STAR exon 1 deletion caused lipoid CAH.Exon 1 substitution does not affect biochemical activity.StAR promoter is responsible for gonadal development.

Physicochemical Changes and Glycation Reaction in Intermediate-Moisture Protein-Sugar Foods with and without Addition of Resveratrol during Storage.

PubMed

Sheng, Zhanwu; Gu, Mantun; Hao, Wangjun; Shen, Yixiao; Zhang, Weimin; Zheng, Lili; Ai, Binling; Zheng, Xiaoyan; Xu, Zhimin

2016-06-22

An intermediate-moisture food (IMF) model consisting of whey protein isolate and glucose and an IMF model fortified with resveratrol were used to study the effect of resveratrol on physicochemical changes and glycation of protein-sugar-rich foods during storage. The water activity (aw) of the storage was controlled at 0.75 or 0.56. The browning rate or hardness of fortified IMFs was significantly lower than that of IMFs after 45-day storage. The rate of Maillard reaction in the samples stored at aw 0.56 was higher than that of samples stored at aw 0.75. The fortified IMFs had lower levels of AGEs (advanced glycation end products), CML (N(ε)-(carboxymethyl)-l-lysine), and insoluble protein during storage. The inhibition capability of resveratrol against glycation was also confirmed by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), liquid chromatography mass spectrometry (LC-MS), and Fourier transform infrared spectroscopy (FTIR) analysis to monitor glycated proteins and protein aggregation in the samples. The results of this study suggested that resveratrol could be used as an inhibitor to reduce the formation of undesirable AGEs and other Maillard reaction products in foods during storage.

De novo disruption of promoter and exon 1 of STAR gene reveals essential role for gonadal development

PubMed Central

Piya, Anil; Kaur, Jasmeet; Rice, Alan M

2017-01-01

Summary Cholesterol transport into the mitochondria is required for synthesis of the first steroid, pregnenolone. Cholesterol is transported by the steroidogenic acute regulatory protein (STAR), which acts at the outer mitochondrial membrane prior to its import. Mutations in the STAR protein result in lipoid congenital adrenal hyperplasia (CAH). Although the STAR protein consists of seven exons, biochemical analysis in nonsteroidogenic COS-1 cells showed that the first two were not essential for pregnenolone synthesis. Here, we present a patient with ambiguous genitalia, salt-lossing crisis within two weeks after birth and low cortisol levels. Sequence analysis of the STAR, including the exon–intron boundaries, showed the complete deletion of exon 1 as well as more than 50 nucleotides upstream of STAR promoter. Mitochondrial protein import with the translated protein through synthesis cassette of the mutant STAR lacking exon 1 showed protein translation, but it is less likely to have synthesized without a promoter in our patient. Thus, a full-length STAR gene is necessary for physiological mitochondrial cholesterol transport in vivo. Learning points: STAR exon 1 deletion caused lipoid CAH. Exon 1 substitution does not affect biochemical activity. StAR promoter is responsible for gonadal development. PMID:28458886

Influence of protein intake from haem and non-haem animals and plant origin on inflammatory biomarkers among apparently-healthy adults in Greece.

PubMed

Vallianou, Natalia G; Bountziouka, Vassiliki P; Georgousopoulou, Ekavi; Evangelopoulos, Angelos A; Bonou, Maria S; Vogiatzakis, Evangelos D; Barbetseas, John D; Avgerinos, Peter C; Panagiotakos, Demosthenes B

2013-12-01

Intake of different types of protein may be associated with differences in biomarkers among various populations. This work investigated the influence of protein intake from haem and non-haem animals as well as protein from plants on haematological and biochemical parameters in inflammation among apparently-healthy adults living in Greece, a Mediterranean country. Four hundred and ninety apparently-healthy subjects (46 +/- 16 years, 40% men), who consecutively visited Polykliniki General Hospital for routine examinations, voluntarily agreed to participate in the study (participation rate 85%). Demographic, anthropometric and lifestyle characteristics were recorded. Participants completed a valid, semi-quantitative food frequency questionnaire. Protein intake was classified into three sources: protein from haem animals, protein from non-haem animals, and protein from plant origin. Fasting blood samples were taken from all participants; uric acid, creatinine, lipids, cystatin C, haptoglobin, haemoglobin, haematocrit, iron, ferritin, white blood cells, monocytes, platelets, and C-reactive protein were measured. Protein intake from only haem animals was associated with increased haemoglobin and haematocrit levels (p < 0.05) whereas intake of protein from non-haem animals and plant origin was not associated with the investigated haematological and biochemical markers of low-grade chronic inflammation when lifestyle factors and overall dietary habits were taken into account. Intake of protein from only haem animals seems to be consistently associated with haematological markers. The confounding role of dietary habits and lifestyle variables on the tested parameters deserves further attention in future research.

The G-protein alpha-subunit gene CGA1 is involved in regulation of resistance to heat and osmotic stress in Chlamydomonas reinhardtii.

PubMed

Lee, C S; Ahn, W; Choi, Y E

2017-02-28

In eukaryotic cells, many important functions of specific G-proteins have been identified, but microalgal G-proteins are poorly studied. In this work, we characterized a gene (CGA1) encoding the G-protein α-subunit in Chlamydomonas reinhardtii. Independent knockdown mutants of CGA1 were generated via RNA interference (RNAi). CGA1 expression levels were consistently and significantly reduced in both independent CGA1 mutant cell lines (cga1). Both cga1 mutants had a higher survival rate at 35°C in comparison with the wild type. This stronger resistance of the cga1 mutants became more evident during simultaneous exposure to heat and osmotic stress. The stronger resistance of the CGA1 knockdown mutants to the two stressors was accompanied with significant morphological alterations-both cell size and cell wall thickness were different from those of the wild type. This finding supports the roles of CGA1 in C. reinhardtii morphology in response to stressors. To further understand biochemical mechanisms of the CGA1-mediated resistance, we thoroughly analyzed the level of reactive oxygen species (ROS) and the expression of several heat shock proteins or MAP kinase genes as possible downstream effectors of CGA1. Our data clearly indicated that CGA1 is implicated in the regulation of resistance to heat or osmotic stress in C. reinhardtii via HSP70A and MAPK6. Because the G-protein α-subunit is highly conserved across microalgal species, our results should facilitate future biotechnological applications of microalgae under extreme environmental conditions.

Disclosure of Selective Advantages in the “modern” Sublineage of the Mycobacterium tuberculosis Beijing Genotype Family by Quantitative Proteomics*

PubMed Central

de Keijzer, Jeroen; de Haas, Petra E.; de Ru, Arnoud H.; van Veelen, Peter A.; van Soolingen, Dick

2014-01-01

The Mycobacterium tuberculosis Beijing genotype, consisting of the more ancient (atypical) and modern (typical) emerging sublineage, is one of the most prevalent and genetically conserved genotype families and has often been associated with multidrug resistance. In this study, we employed a 2D-LC-FTICR MS approach, combined with dimethylation of tryptic peptides, to systematically compare protein abundance levels of ancient and modern Beijing strains and identify differences that could be associated with successful spread of the modern sublineage. The data is available via ProteomeXchange using the identifier PXD000931. Despite the highly uniform protein abundance ratios in both sublineages, we identified four proteins as differentially regulated between both sublineages, which could explain the apparent increased adaptation of the modern Beijing strains. These proteins are; Rv0450c/MmpL4, Rv1269c, Rv3137, and Rv3283/sseA. Transcriptional and functional analysis of these proteins in a large cohort of 29 Beijing strains showed that the mRNA levels of Rv0450c/MmpL4 are significantly higher in modern Beijing strains, whereas we also provide evidence that Rv3283/sseA is less abundant in the modern Beijing sublineage. Our findings provide a possible explanation for the increased virulence and success of the modern Beijing sublineage. In addition, in the established dataset of 1817 proteins, we demonstrate the pre-existence of several, possibly unique, antibiotic efflux pumps in the proteome of the Beijing strains. This may reflect an increased ability of Beijing strains to escape exposure to antituberculosis drugs. PMID:25022876

Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

PubMed Central

Ng, Daphne T. W.

2013-01-01

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts. PMID:23124224

Engineering signal peptides for enhanced protein secretion from Lactococcus lactis.

PubMed

Ng, Daphne T W; Sarkar, Casim A

2013-01-01

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

Differential regulation of cyclo-oxygenase-2 and 5-lipoxygenase-activating protein (FLAP) expression by glucocorticoids in monocytic cells.

PubMed

Goppelt-Struebe, M; Schaefer, D; Habenicht, A J

1997-10-01

1. The objective of the present study was to determine the effects of dexamethasone on key constituents of prostaglandin and leukotriene biosynthesis, cyclo-oxygenase-2 (COX-2) and 5-lipoxygenase activating protein (FLAP). The human monocytic cell line THP-1 was used as a model system. mRNA and protein levels of COX-2 and FLAP were determined by Northern and Western blot analyses, respectively. 2. Low levels of COX-2 and FLAP mRNA were expressed in undifferentiated THP-1 cells, but were induced upon differentiation of the cells along the monocytic pathway by treatment with phorbol ester (TPA, 5 nM). Maximal expression was observed after two days. 3. Coincubation of the undifferentiated cells with dexamethasone (10(-9) - 10(-6) M) and phorbol ester prevented induction of COX-2 mRNA, but did not affect the induction of FLAP mRNA. 4. Dexamethasone downregulated COX-2 mRNA and protein in differentiated, monocyte-like THP-1 cells. In contrast, FLAP mRNA and protein were upregulated by dexamethasone in differentiated THP-1 cells. After 24 h, FLAP mRNA levels were increased more than 2 fold. Dexamethasone did not change 5-lipoxygenase mRNA expression. 5. Release of prostaglandin E2 (PGE2) and peptidoleukotrienes was determined in cell culture supernatants of differentiated THP-1 cells by ELISA. Calcium ionophore-dependent PGE2 synthesis was associated with COX-2 expression, whereas COX-1 and COX-2 seemed to participate in arachidonic acid-dependent PGE2 synthesis. Very low levels of peptidoleukotrienes were released from differentiated THP-1 cells upon incubation with ionophore. Treatment with dexamethasone did not significantly affect leukotriene release. 6. These data provide evidence that prostaglandin synthesis is consistently downregulated by glucocorticoids. However, the glucocorticoid-mediated induction of FLAP may provide a mechanism to maintain leukotriene biosynthesis through more efficient transfer of arachidonic acid to the 5-lipoxygenase reaction, in spite of inhibitory effects on other enzymes of the biosynthetic pathway.

Insulin-sensitizing effect of rosiglitazone (BRL-49653) by regulation of glucose transporters in muscle and fat of Zucker rats.

PubMed

Kramer, D; Shapiro, R; Adler, A; Bush, E; Rondinone, C M

2001-11-01

Thiazolidinediones (TZDs), a class of antidiabetic agents, are specific agonists of peroxisome proliferator activator receptor (PPARgamma). However, their mechanisms of action, and the in vivo target tissues that mediate insulin sensitization are not well understood. The aim of this study was to investigate the role of glucose transporters (GLUT-1 and GLUT-4) in the TZD insulin-sensitizer action. The effects of rosiglitazone treatment were studied using Zucker (fa/fa) rats after 7 days of oral dosing (3.6 mg/kg/d). Rosiglitazone lowered (approximate 80%) basal plasma insulin levels in obese rats and substantially corrected (approximately 50%) insulin resistance based upon results from hyperinsulinemic euglycemic clamp studies. GLUT-4 protein levels were reduced (approximately 75%) in adipose tissue of obese rats and treatment with rosiglitazone normalized them. Interestingly, GLUT-1 protein content was increased in adipose tissue ( thick approximate 150%) and skeletal muscle (approximately 50%) of obese rats and treatment with rosiglitazone increased it even more by 5.5-fold in fat and by 2.5-fold in muscle. Consistent with these results, basal (GLUT-1-mediated) transport rate of 3-O-methyl-D-glucose into isolated epitrochlearis muscle was elevated in response to rosiglitazone. Incubation of fully differentiated 3T3-L1 adipocytes with the drug for 7 days increased the levels of GLUT-1 protein, but did not affect GLUT-4 levels. In conclusion, rosiglitazone may improve insulin resistance in vivo by normalizing GLUT-4 protein content in adipose tissue and increasing GLUT-1 in skeletal muscle and fat. While the drug has a direct effect on GLUT-1 protein expression in vitro without a direct effect on GLUT-4 suggests that direct and indirect effects of rosiglitazone on glucose transporters may have an important role in improving insulin resistance in vivo. Copyright 2001 by W.B. Saunders Company

Striatal-enriched protein tyrosine phosphatase modulates nociception: evidence from genetic deletion and pharmacological inhibition

PubMed Central

Azkona, Garikoitz; Saavedra, Ana; Aira, Zigor; Aluja, David; Xifró, Xavier; Baguley, Tyler; Alberch, Jordi; Ellman, Jonathan A.; Lombroso, Paul J.; Azkue, Jon J.; Pérez-Navarro, Esther

2016-01-01

The information from nociceptors is processed in the dorsal horn of the spinal cord by complex circuits involving excitatory and inhibitory interneurons. It is well documented that GluN2B and ERK1/2 phosphorylation contributes to central sensitization. Striatal-enriched protein tyrosine phosphatase (STEP) dephosphorylates GluN2B and ERK1/2, promoting internalization of GluN2B and inactivation of ERK1/2. The activity of STEP was modulated by genetic (STEP knockout mice) and pharmacological (recently synthesized STEP inhibitor, TC-2153) approaches. STEP61 protein levels in the lumbar spinal cord were determined in male and female mice of different ages. Inflammatory pain was induced by complete Freund’s adjuvant injection. Behavioral tests, immunoblotting, and electrophysiology were used to analyze the effect of STEP on nociception. Our results show that both genetic deletion and pharmacological inhibition of STEP induced thermal hyperalgesia and mechanical allodynia, which were accompanied by increased pGluN2BTyr1472 and pERK1/2Thr202/Tyr204 levels in the lumbar spinal cord. Striatal-enriched protein tyrosine phosphatase heterozygous and knockout mice presented a similar phenotype. Furthermore, electrophysiological experiments showed that TC-2153 increased C fiber-evoked spinal field potentials. Interestingly, we found that STEP61 protein levels in the lumbar spinal cord inversely correlated with thermal hyperalgesia associated with age and female gender in mice. Consistently, STEP knockout mice failed to show age-related thermal hyperalgesia, although gender-related differences were preserved. Moreover, in a model of inflammatory pain, hyperalgesia was associated with increased phosphorylation-mediated STEP61 inactivation and increased pGluN2BTyr1472 and pERK1/2Thr202/Tyr204 levels in the lumbar spinal cord. Collectively, the present results underscore an important role of spinal STEP activity in the modulation of nociception. PMID:26270590

Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

PubMed Central

Mayer-Jaekel, R E; Baumgartner, S; Bilbe, G; Ohkura, H; Glover, D M; Hemmings, B A

1992-01-01

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner. Images PMID:1320961

Inference of RNA decay rate from transcriptional profiling highlights the regulatory programs of Alzheimer's disease.

PubMed

Alkallas, Rached; Fish, Lisa; Goodarzi, Hani; Najafabadi, Hamed S

2017-10-13

The abundance of mRNA is mainly determined by the rates of RNA transcription and decay. Here, we present a method for unbiased estimation of differential mRNA decay rate from RNA-sequencing data by modeling the kinetics of mRNA metabolism. We show that in all primary human tissues tested, and particularly in the central nervous system, many pathways are regulated at the mRNA stability level. We present a parsimonious regulatory model consisting of two RNA-binding proteins and four microRNAs that modulate the mRNA stability landscape of the brain, which suggests a new link between RBFOX proteins and Alzheimer's disease. We show that downregulation of RBFOX1 leads to destabilization of mRNAs encoding for synaptic transmission proteins, which may contribute to the loss of synaptic function in Alzheimer's disease. RBFOX1 downregulation is more likely to occur in older and female individuals, consistent with the association of Alzheimer's disease with age and gender."mRNA abundance is determined by the rates of transcription and decay. Here, the authors propose a method for estimating the rate of differential mRNA decay from RNA-seq data and model mRNA stability in the brain, suggesting a link between mRNA stability and Alzheimer's disease."

Comparative analysis of barophily-related amino acid content in protein domains of Pyrococcus abyssi and Pyrococcus furiosus.

PubMed

Yafremava, Liudmila S; Di Giulio, Massimo; Caetano-Anollés, Gustavo

2013-01-01

Amino acid substitution patterns between the nonbarophilic Pyrococcus furiosus and its barophilic relative P. abyssi confirm that hydrostatic pressure asymmetry indices reflect the extent to which amino acids are preferred by barophilic archaeal organisms. Substitution patterns in entire protein sequences, shared protein domains defined at fold superfamily level, domains in homologous sequence pairs, and domains of very ancient and very recent origin now provide further clues about the environment that led to the genetic code and diversified life. The pyrococcal proteomes are very similar and share a very early ancestor. Relative amino acid abundance analyses showed that biases in the use of amino acids are due to their shared fold superfamilies. Within these repertoires, only two of the five amino acids that are preferentially barophilic, aspartic acid and arginine, displayed this preference significantly and consistently across structure and in domains appearing in the ancestor. The more primordial asparagine, lysine and threonine displayed a consistent preference for nonbarophily across structure and in the ancestor. Since barophilic preferences are already evident in ancient domains that are at least ~3 billion year old, we conclude that barophily is a very ancient trait that unfolded concurrently with genetic idiosyncrasies in convergence towards a universal code.

Experimental and computational laser tissue welding using a protein patch.

PubMed

Small, W; Heredia, N J; Maitland, D J; Eder, D C; Celliers, P M; Da Silva, L B; London, R A; Matthews, D L

1998-01-01

An in vitro study of laser tissue welding mediated with a dye-enhanced protein patch was conducted. Fresh sections of porcine aorta were used for the experiments. Arteriotomies were treated using an indocyanine green dye-enhanced collagen patch activated by an 805-nm continuous-wave fiber-delivered diode laser. Temperature histories of the surface of the weld site were obtained using a hollow glass optical fiber-based two-color infrared thermometer. The experimental effort was complemented by simulations with the LATIS (LAser-TISsue) computer code, which uses coupled Monte Carlo, thermal transport, and mass transport models. Comparison of simulated and experimental thermal data indicated that evaporative cooling clamped the surface temperature of the weld site below 100 °C. For fluences of approximately 200 J/cm2, peak surface temperatures averaged 74°C and acute burst strengths consistently exceeded 0.14×106 dyn/cm (hoop tension). The combination of experimental and simulation results showed that the inclusion of water transport and evaporative losses in the computer code has a significant impact on the thermal distributions and hydration levels throughout the tissue volume. The solid-matrix protein patch provided a means of controllable energy delivery and yielded consistently strong welds. © 1998 Society of Photo-Optical Instrumentation Engineers.

Unique spatial and cellular expression patterns of Hoxa5, Hoxb4 and Hoxb6 proteins in normal developing murine lung are modified in pulmonary hypoplasia

PubMed Central

Volpe, MaryAnn Vitoria; Wang, Karen Ting Wai; Nielsen, Heber Carl; Chinoy, Mala Romeshchandra

2009-01-01

Background Hox transcription factors modulate signaling pathways controlling organ morphogenesis and maintain cell fate and differentiation in adults. Retinoid signaling, key in regulating Hox expression, is altered in pulmonary hypoplasia. Information on pattern-specific expression of Hox proteins in normal lung development and in pulmonary hypoplasia is minimal. Our objective was to determine how pulmonary hypoplasia alters temporal, spatial and cellular expression of Hoxa5, Hoxb4 and Hoxb6 proteins compared to normal lung development. Methods Temporal, spatial and cellular Hoxa5, Hoxb4 and Hoxb6 expression was studied in normal (untreated) and nitrofen-induced hypoplastic (NT-PH) lungs from gestational day 13.5, 16, 19 fetuses and neonates using western blot and immunohistochemistry. Results Modification of protein levels and spatial and cellular Hox expression patterns in NT-PH lungs was consistent with delayed lung development. Distinct protein isoforms were detected for each Hox protein. Expression levels of the Hoxa5 and Hoxb6 isoforms changed with development and further in NT-PH lungs. Compared to normal lungs, Gd19 and neonatal NT-PH lungs had decreased Hoxb6 and increased Hoxa5 and Hoxb4. Hoxa5 cellular localization changed from mesenchyme to epithelia earlier in normal lungs. Hoxb4 was expressed in mesenchyme and epithelial cells throughout development. Hoxb6 remained mainly in mesenchymal cells around distal airways. Conclusions Unique spatial and cellular expression of Hoxa5, Hoxb4 and Hoxb6 participates in branching morphogenesis and terminal sac formation. Altered Hox protein temporal and cellular balance of expression either contributes to pulmonary hypoplasia or functions as a compensatory mechanism attempting to correct abnormal lung development and maturation in this condition. PMID:18553509

Adsorption of intrinsically disordered barnacle adhesive proteins on silica surface

NASA Astrophysics Data System (ADS)

Wang, Xiaoqiang; Wang, Chao; Xu, Baomei; Wei, Junting; Xiao, Yang; Huang, Fang

2018-01-01

The adsorption of recombinant barnacle proteins Bacp19k and Mrcp19k on hydrophilic silica surface was characterized by spectroscopic ellipsometry in artificial seawater (pH = 8.2). They are homologous adhesive proteins destined for underwater adhesion but bear opposite net charges in seawater. As assessed with their primary and secondary structures, both proteins are intrinsically disordered and thus distinct from globular proteins that have dominated research in the field. Different from Mrcp19k, higher initial rate and adsorbed amount were obtained via curve fitting for Bacp19k in kinetic studies, due to favorable charge interactions with silica surface. The good fitting with the same dynamic model also indicates the formation of monolayer coverage in both cases. The two adsorption isotherms of Bacp19k and Mrcp19k are different in the initial change and maximum adsorption level, indicating different protein-surface affinities and charge interactions. Each isotherm fits the Langmuir model well, which is commonly used to describe monolayer adsorption, thus consistent with the predication from kinetic fitting. To further examine the effect of electrostatic interaction on the adsorption, the isotherm of the 1:1 mixture of Bacp19k and Mrcp19k was also constructed, which showed a higher correlation fit for Jovanovic than for Langmuir model. The presence of electrostatic attraction between Bacp19k and Mrcp19k deviated from one of the required conditions for Langmuir behavior, which may also result in the highest coadsorption level but slowest initial change among the three isotherms. The surface state of the adhesive proteins and the change with adsorption time were also examined by atomic force microscopy. The results thus obtained are in good agreement with the corresponding ellipsometric measurement.

Association between Twist and multidrug resistance gene-associated proteins in Taxol®-resistant MCF-7 cells and a 293 cell model of Twist overexpression.

PubMed

Wang, Li; Tan, Rui-Zhi; Zhang, Zhi-Xia; Yin, Rui; Zhang, Yong-Liang; Cui, Wei-Jia; He, Tao

2018-01-01

Multidrug resistance (MDR) severely limits the effectiveness of chemotherapy. Previous studies have identified Twist as a key factor of acquired MDR in breast, gastric and prostate cancer. However, the underlying mechanisms of action of Twist in MDR remain unclear. In the present study, the expression levels of MDR-associated proteins, including lung resistance-related protein (LRP), topoisomerase IIα (TOPO IIα), MDR-associated protein (MRP) and P-glycoprotein (P-gp), and the expression of Twist in cancerous tissues and pericancerous tissues of human breast cancer, were examined. In order to simulate Taxol ® resistance in cells, a Taxol ® -resistant human mammary adenocarcinoma cell subline (MCF-7/Taxol ® ) was established by repeatedly exposing MCF-7 cells to high concentrations of Taxol ® (up to 15 µg/ml). Twist was also overexpressed in 293 cells by transfecting this cell line with pcDNA5/FRT/TO vector containing full-length hTwist cDNA to explore the dynamic association between Twist and MDR gene-associated proteins. It was identified that the expression levels of Twist, TOPO IIα, MRP and P-gp were upregulated and LRP was downregulated in human breast cancer tissues, which was consistent with the expression of these proteins in the Taxol ® -resistant MCF-7 cell model. Notably, the overexpression of Twist in 293 cells increased the resistance to Taxol ® , Trichostatin A and 5-fluorouracil, and also upregulated the expression of MRP and P-gp. Taken together, these data demonstrated that Twist may promote drug resistance in cells and cancer tissues through regulating the expression of MDR gene-associated proteins, which may assist in understanding the mechanisms of action of Twist in drug resistance.

Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation*

PubMed Central

Neumayer, Gernot; Helfricht, Angela; Shim, Su Yeon; Le, Hoa Thi; Lundin, Cecilia; Belzil, Camille; Chansard, Mathieu; Yu, Yaping; Lees-Miller, Susan P.; Gruss, Oliver J.; van Attikum, Haico; Helleday, Thomas; Nguyen, Minh Dang

2012-01-01

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G0 and G1 phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage. PMID:23045526

Circulating Adipokines and Vascular Function: Cross-Sectional Associations in a Community-Based Cohort.

PubMed

Zachariah, Justin P; Hwang, Susan; Hamburg, Naomi M; Benjamin, Emelia J; Larson, Martin G; Levy, Daniel; Vita, Joseph A; Sullivan, Lisa M; Mitchell, Gary F; Vasan, Ramachandran S

2016-02-01

Adipokines may be potential mediators of the association between excess adiposity and vascular dysfunction. We assessed the cross-sectional associations of circulating adipokines with vascular stiffness in a community-based cohort of younger adults. We related circulating concentrations of leptin and leptin receptor, adiponectin, retinol-binding protein 4, and fatty acid-binding protein 4 to vascular stiffness measured by arterial tonometry in 3505 Framingham Third Generation cohort participants free of cardiovascular disease (mean age 40 years, 53% women). Separate regression models estimated the relations of each adipokine to mean arterial pressure and aortic stiffness, as carotid femoral pulse wave velocity, adjusting for age, sex, smoking, heart rate, height, antihypertensive treatment, total and high-density lipoprotein cholesterol, diabetes mellitus, alcohol consumption, estimated glomerular filtration rate, glucose, and C-reactive protein. Models evaluating aortic stiffness also were adjusted for mean arterial pressure. Mean arterial pressure was positively associated with blood retinol-binding protein 4, fatty acid-binding protein 4, and leptin concentrations (all P<0.001) and inversely with adiponectin (P=0.002). In fully adjusted models, mean arterial pressure was positively associated with retinol-binding protein 4 and leptin receptor levels (P<0.002 both). In fully adjusted models, aortic stiffness was positively associated with fatty acid-binding protein 4 concentrations (P=0.02), but inversely with leptin and leptin receptor levels (P≤0.03 both). In our large community-based sample, circulating concentrations of select adipokines were associated with vascular stiffness measures, consistent with the hypothesis that adipokines may influence vascular function and may contribute to the relation between obesity and hypertension. © 2015 American Heart Association, Inc.

Chronic copper exposure causes spatial memory impairment, selective loss of hippocampal synaptic proteins, and activation of PKR/eIF2α pathway in mice.

PubMed

Ma, Quan; Ying, Ming; Sui, Xiaojing; Zhang, Huimin; Huang, Haiyan; Yang, Linqing; Huang, Xinfeng; Zhuang, Zhixiong; Liu, Jianjun; Yang, Xifei

2015-01-01

Copper is an essential element for human growth and development; however, excessive intake of copper could contribute to neurotoxicity. Here we show that chronic exposure to copper in drinking water impaired spatial memory with simultaneous selective loss of hippocampal pre-synaptic protein synapsin 1, and post-synaptic density protein (PSD)-93/95 in mice. Copper exposure was shown to elevate the levels of nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampus, two markers of oxidative stress. Concurrently, we also found that copper exposure activated double stranded RNA-dependent protein kinase (PKR) as evidenced by increased ratio of phosphorylated PKR at Thr451 and total PKR and increased the phosphorylation of its downstream signaling molecule eukaryotic initiation factor 2α (eIF2α) at Ser51 in hippocampus. Consistent with activation of PKR/eIF2α signaling pathway which was shown to mediate synaptic deficit and cognitive impairment, the levels of activating transcription factor 4 (ATF-4), a downstream signaling molecule of eIF2α and a repressor of CREB-mediated gene expression, were significantly increased, while the activity of cAMP response elements binding protein (CREB) was inactivated as suggested by decreased phosphorylation of CREB at Ser133 by copper exposure. In addition, the expression of the pro-apoptotic target molecule C/EBP homology protein (CHOP) of ATF-4 was upregulated and hippocampal neuronal apoptosis was induced by copper exposure. Taken together, we propose that chronic copper exposure might cause spatial memory impairment, selective loss of synaptic proteins, and neuronal apoptosis through the mechanisms involving activation of PKR/eIF2α signaling pathway.

Apparent total tract macronutrient digestibility, fecal characteristics, and fecal fermentative end-product concentrations of healthy adult dogs fed bioprocessed soy protein.

PubMed

Beloshapka, A N; de Godoy, M R C; Detweiler, K B; Newcomb, M; Ellegård, K H; Fahey, G C; Swanson, K S

2016-09-01

Animal proteins are commonly used in extruded dog foods. Plant-based proteins have a more consistent nutrient profile than animal sources but may contain antinutritional factors, including trypsin inhibitors and oligosaccharides. Bioprocessed soy protein (SP; HP-300; Hamlet Protein, Inc., Findlay, OH) is a processed soy-based product with low antinutritional factor concentrations and high protein quality. The objective was to evaluate the effects of SP on apparent total tract macronutrient digestibility, fecal characteristics, and fecal fermentative end products. Furthermore, this study aimed to identify if SP can be a replacement for poultry byproduct meal (PBPM) in dog food and determine if there are practical limits to its use. Three palatability experiments were conducted to evaluate 1) 0 vs. 12% SP, 2) 0 vs. 48% SP, and 3) 12 vs. 48% SP. For digestibility, 48 healthy adult Beagle dogs (20 females and 28 males; 3.4 yr mean age and 10.0 kg mean BW) were randomly allotted to 1 of 6 dietary treatments, 0 (control), 4, 8, 12, 24, and 48% SP, in a completely randomized design. All diets were formulated to meet Association of American Feed Control Officials nutrient profiles and contained approximately 30% CP and 16% fat. The treatment period consisted of a 10-d diet adaptation phase followed by a 4-d fresh and total fecal collection phase. The palatability results suggest that of the 3 inclusion levels tested (0, 12, or 48% SP), the best inclusion of SP is 12%, which was preferred over 0 and 48% SP. Digestibility and fecal data were evaluated for linear and quadratic effects using SAS. Stool output (on both an as-is and a DM basis) did not differ from the control except for the 48% SP treatment ( < 0.01). Fecal output per unit food intake differed ( < 0.01) from the control only at the 24 and 48% SP inclusion rates. No significant effects of feeding SP were found on stool consistency scores. Digestibility of DM, OM, and energy did not differ from the control at any inclusion rate, except for a decrease ( < 0.01) at 48% SP. Apparent total tract CP digestibility was not affected by treatment and ranged from 82.9 to 86.2%. Fecal short-chain fatty acid concentrations were greater ( < 0.01) in dogs fed 24 and 48% SP compared with the control. Conversely, branched-chain fatty acid concentrations were lower ( < 0.01) in dogs fed 8 to 48% SP compared with the control. These data suggest that SP is a suitable replacement for PBPM in dog diets up to a 24% inclusion level.

[Surgical therapy of segmental jejunal, primary intestinal lymphangiectasia].

PubMed

Kneist, W; Drescher, D G; Hansen, T; Kreitner, K F; Lang, H

2013-06-01

Primary intestinal lymphangiectasia (PIL) is a protein-losing, exsudative gastroenteropathy causing lymphatic obstruction. Diagnosis depends on clinical examination and histological findings. Conservative treatment modalities include a low-fat diet and enteral nutritional therapy in order to reduce enteric protein loss and to improve fat metabolism. Other treatment options consist of administration of antiplasmin or octreotide to lower lymph flow and secretion. We report on a 58-year-old patient who underwent exploratory laparotomy due to a worsening physical status, recurrent chylaskos and leg oedema under conservative dietary therapy. Intraoperative findings showed a typical PIL of the jejunum about 20 cm distal to the Treitz's ligament. Histological examinations confirmed this diagnosis. One year after segmental small bowel resection (105 cm) with end-to-end anastomosis the patient is healthy, free of symptoms, has gained weight and his serum protein level has increased. Intraabdominal ascites and leg oedema have not reoccurred since.