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Sample records for acute regulatory protein

  1. Mitochondrial fusion and ERK activity regulate steroidogenic acute regulatory protein localization in mitochondria.

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    Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J; Poderoso, Cecilia

    2014-01-01

    The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR.

  2. Steroidogenic acute regulatory protein (StAR), a novel mitochondrial cholesterol transporter.

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    Miller, Walter L

    2007-06-01

    Cholesterol is a vital component of cellular membranes, and is the substrate for biosynthesis of steroids, oxysterols and bile acids. The mechanisms directing the intracellular trafficking of this nearly insoluble molecule have received increased attention through the discovery of the steroidogenic acute regulatory protein (StAR) and similar proteins containing StAR-related lipid transfer (START) domains. StAR can transfer cholesterol between synthetic liposomes in vitro, an activity which appears to correspond to the trans-cytoplasmic transport of cholesterol to mitochondria. However, trans-cytoplasmic cholesterol transport in vivo appears to involve the recently-described protein StarD4, which is expressed in most cells. Steroidogenic cells must also move large amounts of cholesterol from the outer mitochondrial membrane to the first steroidogenic enzyme, which lies on the matrix side of the inner membrane; this action requires StAR. Congenital lipoid adrenal hyperplasia, a rare and severe disorder of human steroidogenesis, results from mutations in StAR, providing a StAR knockout of nature that has provided key insights into its activity. Cell biology experiments show that StAR moves large amounts of cholesterol from the outer to inner mitochondrial membrane, but acts exclusively on the outer membrane. Biophysical data show that only the carboxyl-terminal alpha-helix of StAR interacts with the outer membrane. Spectroscopic data and molecular dynamics simulations show that StAR's interactions with protonated phospholipid head groups on the outer mitochondrial membrane induce a conformational change (molten globule transition) needed for StAR's activity. StAR appears to act in concert with the peripheral benzodiazepine receptor, but the precise itinerary of a cholesterol molecule entering the mitochondrion remains unclear.

  3. Expression of steroidogenic acute regulatory protein and its regulation by interferon-gamma in rat corpus luteum

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The steroidogenic acute regulatory protein (StAR) is the key regulatory protein of steroidogenesis. De novo synthesis of StAR protein is required for intramitochondrial translocation of cholesterol to the cytochrome P450 side chain cleavage enzyme which is located on the matrix side of the inner mitochondrial membrane. This is the rate-limiting step of steroid biosynthesis. Using in situ hybridization and immunohistochemistry we studied StAR expression in various stages of the corpora luteal and its regulation by interferon-gamma (IFNγ) in the adult pseudopregnant rat. The results indicated that expression of StAR in the corpora luteal was correlated with progesteron production and IFNγ was capable of inhibiting its expression.

  4. MAR binding protein SMAR1 favors IL-10 mediated regulatory T cell function in acute colitis

    Energy Technology Data Exchange (ETDEWEB)

    Mirlekar, Bhalchandra; Patil, Sachin [Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411007 (India); Bopanna, Ramanamurthy [Experimental Animal Facility, National Centre for Cell Science, Ganeshkhind, Pune 411007 (India); Chattopadhyay, Samit, E-mail: samit@nccs.res.in [Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411007 (India)

    2015-08-21

    T{sub reg} cells are not only crucial for controlling immune responses to autoantigens but also prevent those directed towards commensal pathogens. Control of effector immune responses by T{sub reg} cells depend on their capacity to accumulate at inflammatory site and accordingly accommodate to inflammatory environment. Till date, the factors associated with maintaining these aspects of T{sub reg} phenotype is not understood properly. Here we have shown that a known nuclear matrix binding protein SMAR1 is selectively expressed more in colonic T{sub reg} cells and is required for their ability to accumulate at inflammatory site and to sustain high levels of Foxp3 and IL-10 expression during acute colitis. Elimination of anti-inflammatory subsets revealed a protective role for IL-10 producing T{sub reg} cells in SMAR1{sup −/−} mice. Moreover, a combined action of Foxp3 and SMAR1 restricts effector cytokine production and enhance the production of IL-10 by colonic T{sub reg} cells that controls acute colitis. This data highlights a critical role of SMAR1 in maintaining T{sub reg} physiology during inflammatory disorders. - Highlights: • SMAR1 is essential to sustain high level of Foxp3 and IL-10 in T{sub reg} cells. • SMAR1{sup −/−} T{sub reg} cells produce pro-inflammatory cytokine IL-17 leads to inflammation. • IL-10 administration can control the inflammation in SMAR1{sup −/−} mice. • Both Foxp3 and SMAR1 maintain T{sub reg} phenotype that controls colitis.

  5. Steroidogenic acute regulatory protein in white sturgeon (Acipenser transmontanus): cDNA cloning, sites of expression and transcript abundance in corticosteroidogenic tissue after an acute stressor.

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    Kusakabe, Makoto; Zuccarelli, Micah D; Nakamura, Ikumi; Young, Graham

    2009-06-01

    The white sturgeon, Acipenser transmontanus, is a primitive bony fish that is recognized as an important emerging species for aquaculture. However, many aspects of its stress and reproductive physiology remain unclear. These processes are controlled by various steroid hormones. In order to investigate the regulation of steroidogenesis associated with acute stress in sturgeon, a cDNA-encoding steroidogenic acute regulatory protein (StAR) was isolated from white sturgeon. The putative amino acid sequence of sturgeon StAR shares high homology (over 60%) with other vertebrates. Phylogenetic analysis grouped sturgeon StAR within Actinopterygii, but it was clearly segregated from teleost StARs. RT-PCR analysis revealed that transcripts were most abundant in yellow corpuscles found throughout the kidney and weaker signals were detected in gonad and kidney. Very weak signals were also detected in brain and spleen by quantitative real-time PCR. In situ hybridization revealed that StAR is expressed in the cells of yellow corpuscles. No significant changes in StAR gene expression were detected in response to an acute handling stress. These results suggest that StAR is highly conserved throughout vertebrates, but the expression of the functional protein during the stress response may be partially regulated post-transcriptionally.

  6. Detection of hCG Responsive Expression of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells

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    Manna Pulak R.

    2004-01-01

    Full Text Available The steroidogenic acute regulatory (StAR protein, a novel mitochondrial protein, is involved in the regulation of steroid hormone biosynthesis through its mediation of the intramitochondrial transport of the steroid substrate, cholesterol, to the cytochrome P450 cholesterol side chain cleavage (P450scc enzyme. The expression of StAR protein is regulated by cAMP-dependent signaling in steroidogenic cells. During the course of our studies in mouse Leydig cells, we employ several methods for studying the regulation of StAR protein expression by human chorionic gonadotropin (hCG. A sensitive quantitative reverse transcription and polymerase chain reaction (RT-PCR was utilized for determining StAR mRNA expression. Stimulation of mLTC-1 mouse Leydig tumor cells with hCG resulted in the coordinate regulation of StAR mRNA expression and progesterone accumulation in a time-response manner. The validity and accuracy of quantitative RT-PCR results in mLTC-1 cells were verified by a competitive PCR approach and were further confirmed in primary cultures of isolated mouse Leydig cells. Immunoblotting studies demonstrated an increase in the levels of the StAR protein in a concentration dependent manner following hCG stimulation in mLTC-1 cells. Northern hybridization analysis revealed three StAR transcripts, all of which were of sufficient size to encode functional StAR protein, and which were coordinately expressed in response to hCG. Collectively, the experimental approaches utilized in the present investigation allow for the demonstration and characterization of hCG mediated regulation of StAR mRNA and StAR protein expression in mouse Leydig cells.

  7. Stimulatory effects of propylthiouracil on pregnenolone production through upregulation of steroidogenic acute regulatory protein expression in rat granulosa cells.

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    Chen, Mei-Chih; Wang, Shyi-Wu; Kan, Shu-Fen; Tsai, Shiow-Chwen; Wu, Yu-Ching; Wang, Paulus S

    2010-12-01

    Propylthiouracil (PTU) is a common and effective clinical medicine for the treatment of hyperthyroidism. Our previous study demonstrated that short-term treatment with PTU inhibits progesterone production in rat granulosa cells. However, our present results indicate that a 16-h treatment with PTU was able to stimulate pregnenolone production in rat granulosa cells, although progesterone production was diminished by PTU through inhibition of 3β-hydroxysteroid dehydrogenase. Notably, we found that PTU treatment enhanced the conversion of cholesterol into pregnenolone, whereas the protein level of the cytochrome P450 side-chain cleavage enzyme (P450scc, which is the enzyme responding to this conversion) was not affected. Interestingly, the levels of steroidogenic acute regulatory protein (StAR) in both total cell lysate and the mitochondrial fraction were significantly increased by PTU treatment. Furthermore, the binding of steroidogenic factor-1 (SF-1) to the StAR promoter region was also enhanced by PTU treatment, which suggests that PTU could upregulate StAR gene expression. In addition to SF-1 regulation, we found that mitogen-activated protein (MAP) kinase kinase activation is an important regulator of PTU-stimulated StAR protein expression, based on the effects of the MEK inhibitor PD98059. In conclusion, these results indicate that PTU plays opposite roles in the production of progesterone and its precursor, pregnenolone. The regulation of negative feedback on speeding the cholesterol transportation and pregnenolone conversion after a 16-h PTU treatment may be the mechanism explaining PTU's inhibition of progesterone production in rat granulosa cells.

  8. The expression of tumor necrosis factor-alpha, its receptors and steroidogenic acute regulatory protein during corpus luteum regression

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    Arfuso Frank

    2008-11-01

    Full Text Available Abstract Background Corpus luteum (CL regression is known to occur as two parts; functional regression when steroidogenesis declines and structural regression when apoptosis is induced. Previous studies suggest this process occurs by the production of luteolytic factors, such as tumour necrosis factor-alpha (TNF-alpha. Methods We examined TNF-alpha, TNF-alpha receptors (TNFR1 and 2 and steroidogenic acute regulatory (StAR protein expression during CL regression in albino Wistar rats. CL from Days 16 and 22 of pregnancy and Day 3 post-partum were examined, in addition CL from Day 16 of pregnancy were cultured in vitro to induce apoptosis. mRNA was quantitated by kinetic RT-PCR and protein expression examined by immunohistochemistry and Western blot analyses. Results TNF-alpha mRNA increased on Day 3 post-partum. TNFR were immunolocalized to luteal cells, and an increase in TNFR2 mRNA observed on Day 3 post-partum whilst no change was detected in TNFR1 mRNA relative to Day 16. StAR protein decreased on Day 3 post-partum and following trophic withdrawal but no change was observed following exogenous TNF-alpha treatment. StAR mRNA decreased on Day 3 post-partum; however, it increased following trophic withdrawal and TNF-alpha treatment in vitro. Conclusion These results demonstrate the existence of TNFR1 and TNFR2 in rat CL and suggest the involvement of TNF-alpha in rat CL regression following parturition. Furthermore, decreased StAR expression over the same time points was consistent with the functional regression of the CL.

  9. Characterization of novel StAR (steroidogenic acute regulatory protein mutations causing non-classic lipoid adrenal hyperplasia.

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    Christa E Flück

    Full Text Available CONTEXT: Steroidogenic acute regulatory protein (StAR is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH. OBJECTIVE: StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported. DESIGN: To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature. SETTING: Collaboration between the University Children's Hospital Bern, Switzerland, and the CIBERER, Hospital Vall d'Hebron, Autonomous University, Barcelona, Spain. PATIENTS: Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age. RESULTS: StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (∼30% and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol. CONCLUSIONS: StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed.

  10. Steroidogenic acute regulatory protein (StAR) overexpression attenuates HFD-induced hepatic steatosis and insulin resistance.

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    Qiu, Yanyan; Sui, Xianxian; Zhan, Yongkun; Xu, Chen; Li, Xiaobo; Ning, Yanxia; Zhi, Xiuling; Yin, Lianhua

    2017-04-01

    Non-alcoholic fatty liver disease (NAFLD) covers a wide spectrum of liver pathology. Intracellular lipid accumulation is the first step in the development and progression of NAFLD. Steroidogenic acute regulatory protein (StAR) plays an important role in the synthesis of bile acid and intracellular lipid homeostasis and cholesterol metabolism. We hypothesize that StAR is involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis. The hypothesis was identified using free fatty acid (FFA)-overloaded NAFLD in vitro model and high-fat diet (HFD)-induced NAFLD mouse model transfected by recombinant adenovirus encoding StAR (StAR). StAR expression was also examined in pathology samples of patients with fatty liver by immunohistochemical staining. We found that the expression level of StAR was reduced in the livers obtained from fatty liver patients and NAFLD mice. Additionally, StAR overexpression decreased the levels of hepatic lipids and maintained the hepatic glucose homeostasis due to the activation of farnesoid x receptor (FXR). StAR overexpression attenuated the impairment of insulin signaling in fatty liver. This protective role of StAR was owing to a reduction of intracellular diacylglycerol levels and the phosphorylation of PKCε. Furthermore, FXR inactivation reversed the observed beneficial effects of StAR. The present study revealed that StAR overexpression can reduce hepatic lipid accumulation, regulate glucose metabolism and attenuate insulin resistance through a mechanism involving the activation of FXR. Our study suggests that StAR may be a potential therapeutic target for NAFLD.

  11. Quercetin and vitamin E attenuate Bonny Light crude oil-induced alterations in testicular apoptosis, stress proteins and steroidogenic acute regulatory protein in Wistar rats.

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    Ebokaiwe, Azubuike P; Mathur, Premendu P; Farombi, Ebenezer O

    2016-10-01

    Studies have shown the reproductive effects of Bonny Light crude oil (BLCO) via the mechanism of oxidative stress and testicular apoptosis. We investigated the protective role of quercetin and vitamin E on BLCO-induced testicular apoptosis. Experimental rats were divided into four groups of four each. Animals were orally administered 2 ml/kg corn oil (control: group 1), BLCO-800 mg/kg body weight + 10 mg/kg quercetin (group 2), BLCO-800 mg/kg body weight + 50 mg/kg vitamin E (group 3) and BLCO-800 mg/kg body weight only (group 4) for 7 d. Protein levels of caspase 3, FasL, NF-kB, steroidogenic acute regulatory protein and stress response proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunofluorescence staining was used to quantify the expression of caspase 3, FasL and NF-kB. Apoptosis was quantified by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Administration of BLCO resulted in a significant increase in the levels of stress response proteins and apoptosis-related proteins by 50% and above after 7 d following BLCO exposure and a concomitant increase in expression of caspase 3, FasL and NF-kB expression by immunofluorescence staining. Apoptosis showed a significant increase in TUNEL positive cells. Co-administration with quercetin or vitamin E reversed BLCO-induced apoptosis and levels of stress protein, relative to control. These findings suggest that quercetin and vitamin E may confer protection against BLCO-induced testicular oxidative stress-related apoptosis.

  12. High-yield soluble expression, purification and characterization of human steroidogenic acute regulatory protein (StAR) fused to a cleavable Maltose-Binding Protein (MBP).

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    Sluchanko, Nikolai N; Tugaeva, Kristina V; Faletrov, Yaroslav V; Levitsky, Dmitrii I

    2016-03-01

    Steroidogenic acute regulatory protein (StAR) is responsible for the rapid delivery of cholesterol to mitochondria where the lipid serves as a source for steroid hormones biosynthesis in adrenals and gonads. Despite many successful investigations, current understanding of the mechanism of StAR action is far from being completely clear. StAR was mostly obtained using denaturation/renaturation or in minor quantities in a soluble form at decreased temperatures that, presumably, limited the possibilities for its consequent detailed exploration. In our hands, existing StAR expression constructs could be bacterially expressed almost exclusively as insoluble forms, even upon decreased expression temperatures and in specific strains of Escherichia coli, and isolated protein tended to aggregate and was difficult to handle. To maximize the yield of soluble protein, optimized StAR sequence encompassing functional domain STARD1 (residues 66-285) was fused to the C-terminus of His-tagged Maltose-Binding Protein (MBP) with the possibility to cleave off the whole tag by 3C protease. The developed protocol of expression and purification comprising of a combination of subtractive immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography allowed us to obtain up to 25 mg/1 L culture of completely soluble StAR protein, which was (i) homogenous according to SDS-PAGE, (ii) gave a single symmetrical peak on a gel-filtration, (iii) showed the characteristic CD spectrum and (iv) pH-dependent ability to bind a fluorescently-labeled cholesterol analogue. We conclude that our strategy provides fully soluble and native StAR protein which in future could be efficiently used for biotechnology and drug discovery aimed at modulation of steroids production.

  13. Potential of acute phase proteins as predictor of postpartum uterine infections during transition period and its regulatory mechanism in dairy cattle

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    A. Manimaran

    2016-01-01

    Full Text Available Among the various systemic reactions against infection or injury, the acute phase response is the cascade of reaction and mostly coordinated by cytokines-mediated acute phase proteins (APPs production. Since APPs are sensitive innate immune molecules, they are useful for early detection of inflammation in bovines and believed to be better discriminators than routine hematological parameters. Therefore, the possibility of using APPs as a diagnostic and prognostic marker of inflammation in major bovine health disorders including postpartum uterine infection has been explored by many workers. In this review, we discussed specifically importance of postpartum uterine infection, the role of energy balance in uterine infections and potential of APPs as a predictor of postpartum uterine infections during the transition period and its regulatory mechanism in dairy cattle.

  14. Human chorionic gonadotropin (hCG) regulates the expression of Steroidogenic acute regulatory protein (STAR)via the ERK1/2 pathway

    Institute of Scientific and Technical Information of China (English)

    WANG Xianzhong; SUN Yan; WU Jianyun; PAN Hongmei; ZHANG Jiahua

    2007-01-01

    It has previously been shown that Human Chorionic Gonadotropin (hCG) can stimulate steroidogenesis in Leydig cells.In the present study,the mechanisms of hCGstimulated steroidogenesis in Leydig cells of immaturated pigs were investigated.It was found that both hCG and 8Br-cAMP could enhance the expression level of both the Steroidogenic acute regulatory protein (STAR) and mRNA,and increase the activity of extracellular signal-regulated kinasel/2 (ERK1/2) significantly depending on stimulating time.However,the effect of 8-Br-cAMP was more significant than that of hCG.While appending the inhibitor of Protein Kinase A (PKA) to Leydig cells in culture,the expression level of StAR protein,mRNA and the activity of ERK1/2 began to drop significantly,but the level of StAR mRNA could still be detectable.While appending the inhibitor of MAPK (PD98059),the expression level of StAR protein and mRNA declined significantly.These results infer that at the beginning of hCG stimulation,hCG increases the level of StAR protein by cAMP-PKA.With prolonged stimulating time,hCG increases the level of StAR protein through cAMP-PKA-ERK1/2.

  15. Expression of steroidogenic acute regulatory protein (StAR) in male American bullfrog (Rana catesbeiana) and preliminary evaluation of the response to TNT.

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    Paden, Norka E; Carr, James A; Kendall, Ronald J; Wages, Mike; Smith, Ernest E

    2010-06-01

    We examined the expression of steroidogenic acute regulatory (StAR) protein mRNA in the American bullfrog (Rana catesbeiana). Primers and probes were designed to obtain a partial sequence of bullfrog StAR cDNA consisting of 349 base pairs. Quantitative PCR analysis of StAR mRNA equivalents was performed in tissues of juvenile and adult bullfrogs. In this study 18S mRNA was used as an internal standard. There were no differences in the expression of 18S RNA among tissues or between age groups. In juvenile males, the rank order for the constitutive levels of StAR was testes>skin>brain>kidneys. In adult males, StAR mRNA equivalent was greatest in testes, followed by kidneys, brain, and skin. In addition, stimulation and induction of testicular StAR by human chorionic gonadotropin significantly increased expression of StAR at 2, 4, and 6h after injection. Preliminary evaluation of 2, 4, 6-trinitrotoluene (TNT) revealed that acute exposure is associated with reduction of StAR mRNA expression. The information provided in this study will be useful for future research on StAR gene expression in amphibian reproductive biology and the development of reproductive biomarkers.

  16. Tlr4-mutant mice are resistant to acute alcohol-induced sterol-regulatory element binding protein activation and hepatic lipid accumulation

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    Zhang, Zhi-Hui; Liu, Xiao-Qian; Zhang, Cheng; He, Wei; Wang, Hua; Chen, Yuan-Hua; Liu, Xiao-Jing; Chen, Xi; Xu, De-Xiang

    2016-01-01

    Previous studies demonstrated that acute alcohol intoxication caused hepatic lipid accumulation. The present study showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic sterol-regulatory element binding protein (SREBP)-1, a transcription factor regulating fatty acid and triglyceride (TG) synthesis, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic Fas, Acc, Scd-1 and Dgat-2, the key genes for fatty acid and TG synthesis, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Additional experiment showed that hepatic MyD88 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic NF-κB was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Moreover, hepatic GSH content was reduced and hepatic MDA level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. In conclusion, Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. PMID:27627966

  17. Mechanisms of action of hormone-sensitive lipase in mouse Leydig cells: its role in the regulation of the steroidogenic acute regulatory protein.

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    Manna, Pulak R; Cohen-Tannoudji, Joëlle; Counis, Raymond; Garner, Charles W; Huhtaniemi, Ilpo; Kraemer, Fredric B; Stocco, Douglas M

    2013-03-22

    Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of cholesteryl esters in steroidogenic tissues and, thus, facilitates cholesterol availability for steroidogenesis. The steroidogenic acute regulatory protein (StAR) controls the rate-limiting step in steroid biosynthesis. However, the modes of action of HSL in the regulation of StAR expression remain obscure. We demonstrate in MA-10 mouse Leydig cells that activation of the protein kinase A (PKA) pathway, by a cAMP analog Bt2cAMP, enhanced expression of HSL and its phosphorylation (P) at Ser-660 and Ser-563, but not at Ser-565, concomitant with increased HSL activity. Phosphorylation and activation of HSL coincided with increases in StAR, P-StAR (Ser-194), and progesterone levels. Inhibition of HSL activity by CAY10499 effectively suppressed Bt2cAMP-induced StAR expression and progesterone synthesis. Targeted silencing of endogenous HSL, with siRNAs, resulted in increased cholesteryl ester levels and decreased cholesterol content in MA-10 cells. Depletion of HSL affected lipoprotein-derived cellular cholesterol influx, diminished the supply of cholesterol to the mitochondria, and resulted in the repression of StAR and P-StAR levels. Cells overexpressing HSL increased the efficacy of liver X receptor (LXR) ligands on StAR expression and steroid synthesis, suggesting HSL-mediated steroidogenesis entails enhanced oxysterol production. Conversely, cells deficient in LXRs exhibited decreased HSL responsiveness. Furthermore, an increase in HSL was correlated with the LXR target genes, steroid receptor element-binding protein 1c and ATP binding cassette transporter A1, demonstrating HSL-dependent regulation of steroidogenesis predominantly involves LXR signaling. LXRs interact/cooperate with RXRs and result in the activation of StAR gene transcription. These findings provide novel insight and demonstrate the molecular events by which HSL acts to drive cAMP/PKA-mediated regulation of StAR expression and

  18. Upregulation of Steroidogenic Acute Regulatory Protein by Hypoxia Stimulates Aldosterone Synthesis in Pulmonary Artery Endothelial Cells to Promote Pulmonary Vascular Fibrosis

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    Maron, Bradley A.; Oldham, William M.; Chan, Stephen Y.; Vargas, Sara O.; Arons, Elena; Zhang, Ying-Yi; Loscalzo, Joseph; Leopold, Jane A.

    2014-01-01

    Background The molecular mechanism(s) regulating hypoxia-induced vascular fibrosis are unresolved. Hyperaldosteronism correlates positively with vascular remodeling in pulmonary arterial hypertension (PAH), suggesting that aldosterone may contribute to the pulmonary vasculopathy of hypoxia. The hypoxia-sensitive transcription factors c-Fos/c-Jun regulate steroidogenic acute regulatory protein (StAR), which facilitates the rate-limiting step of aldosterone steroidogenesis. We hypothesized that c-Fos/c-Jun upregulation by hypoxia activates StAR-dependent aldosterone synthesis in human pulmonary artery endothelial cells (HPAECs) to promote vascular fibrosis in PAH. Methods and Results Patients with PAH, rats with Sugen/hypoxia-PAH, and mice exposed to chronic hypoxia expressed increased StAR in remodeled pulmonary arterioles, providing a basis for investigating hypoxia-StAR signaling in HPAECs. Hypoxia (2.0% FiO2) increased aldosterone levels selectively in HPAECs, which was confirmed by liquid chromatography-mass spectrometry. Increased aldosterone by hypoxia resulted from enhanced c-Fos/c-Jun binding to the proximal activator protein (AP-1) site of the StAR promoter in HPAECs, which increased StAR expression and activity. In HPAECs transfected with StAR-siRNA or treated with the AP-1 inhibitor, SR-11302, hypoxia failed to increase aldosterone, confirming that aldosterone biosynthesis required StAR activation by c-Fos/c-Jun. The functional consequences of aldosterone were confirmed by pharmacological inhibition of the mineralocorticoid receptor with spironolactone or eplerenone, which attenuated hypoxia-induced upregulation of the fibrogenic protein connective tissue growth factor and collagen III in vitro, and decreased pulmonary vascular fibrosis to improve pulmonary hypertension in Conclusions Our findings identify autonomous aldosterone synthesis in HPAECs due to hypoxia-mediated upregulation of StAR as a novel molecular mechanism that promotes pulmonary vascular

  19. Inherited complement regulatory protein deficiency predisposes to human disease in acute injury and chronic inflammatory statesthe examples of vascular damage in atypical hemolytic uremic syndrome and debris accumulation in age-related macular degeneration.

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    Richards, Anna; Kavanagh, David; Atkinson, John P

    2007-01-01

    In this chapter, we examine the role of complement regulatory activity in atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration (AMD). These diseases are representative of two distinct types of complement-mediated injury, one being acute and self-limited, the other reflecting accumulation of chronic damage. Neither condition was previously thought to have a pathologic relationship to the immune system. However, alterations in complement regulatory protein genes have now been identified as major predisposing factors for the development of both diseases. In aHUS, heterozygous mutations leading to haploinsufficiency and function-altering polymorphisms in complement regulators have been identified, while in AMD, polymorphic haplotypes in complement genes are associated with development of disease. The basic premise is that a loss of function in a plasma or membrane inhibitor of the alternative complement pathway allows for excessive activation of complement on the endothelium of the kidney in aHUS and on retinal debris in AMD. These associations have much to teach us about the host's innate immune response to acute injury and to chronic debris deposition. We all experience cellular injury and, if we live long enough, will deposit debris in blood vessel walls (atherosclerosis leading to heart attacks and strokes), the brain (amyloid proteins leading to Alzheimer's disease), and retina (lipofuscin pigments leading to AMD). These are three common causes of morbidity and mortality in the developed world. The clinical, genetic, and immunopathologic understandings derived from the two examples of aHUS and AMD may illustrate what to anticipate in related conditions. They highlight how a powerful recognition and effector system, the alternative complement pathway, reacts to altered self. A response to acute injury or chronic debris accumulation must be appropriately balanced. In either case, too much activation or too little regulation promotes

  20. Relationship between changes in mRNAs of the genes encoding steroidogenic acute regulatory protein and P450 cholesterol side chain cleavage in head kidney and plasma levels of cortisol in response to different kinds of acute stress in the rainbow trout (Oncorhynchus mykiss).

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    Geslin, Malika; Auperin, Benoit

    2004-01-01

    In this study, the expression of several genes involved in cortisol synthesis in head kidneys, the site of cortisol production, and in the rainbow trout (Oncorhynchus mykiss) was examined in response to two different acute stressors and an acute ACTH treatment. mRNAs levels of the "steroidogenic acute regulatory" (StAR) sterol transport protein, which transports cholesterol to the inner mitochondrial membrane as well as cytochrome P450 cholesterol side chain cleavage (P450(SCC)) were determined in head kidney (containing the interrenal tissue). In one experiment, we also quantified 3-beta-hydroxysteroid dehydrogenase (3B-HSD) and cytochrome P450(11beta) (11B-H) mRNAs. The presence of these four transcripts in the head kidney was confirmed by Northern blot analysis. For each stress condition, mRNA levels were quantified by quantitative or real-time RT-PCR. The results of these two methods were highly correlated. An acute stress induced by capture, short confinement (2min), and anesthesia (3min) resulted in significant elevation of plasma cortisol (30-fold higher than controls) and an increase in levels of StAR and P450(SCC) mRNAs 3h post-stress. When fish were submitted to an acute stress caused by 5min of chase with a net in a tank, plasma cortisol reached a peak within 1h, but after 3h, levels were only 5-fold higher in stressed trout than in controls and no variations in the expression of StAR, P450(SCC), 3B-HSD, and 11B-H were observed whatever the time post-stress. One hour after acute ACTH stimulation (5IU/kg), plasma cortisol level was 4-fold higher than in control trout and no changes in StAR and P450(SCC) mRNAs levels were detected. The data suggest that the high levels of cortisol after stress need an activation of genes involved in cortisol synthesis, but lower levels do not. Futhermore, under these three test conditions, we always found a strong positive correlation between mRNA levels of StAR and P450(SCC), in contrast to what has been described in

  1. Review: Acute phase reaction and acute phase proteins

    Institute of Scientific and Technical Information of China (English)

    GRUYS E.; TOUSSAINT M.J.M.; NIEWOLD T.A.; KOOPMANS S.J.

    2005-01-01

    A review of the systemic acute phase reaction with major cytokines involved, and the hepatic metabolic changes,negative and positive acute phase proteins (APPs) with function and associated pathology is given. It appears that APPs represent appropriate analytes for assessment of animal health. Whereas they represent non-specific markers as biological effect reactants,they can be used for assessing nutritional deficits and reactive processes, especially when positive and negative acute phase variables are combined in an index. When such acute phase index is applied to separate healthy animals from animals with some disease, much better results are obtained than with single analytes and statistically acceptable results for culling individual animals may be reached.Unfortunately at present no cheap, comprehensive and easy to use system is available for assessing various acute phase proteins in serum or blood samples at the same time. Protein microarray or fluid phase microchip technology may satisfy this need;and permit simultaneous analysis of numerous analytes in the same small volume sample and enable integration of information derived from systemic reactivity and nutrition with disease specific variables. Applying such technology may help to solve health problems in various countries not only in animal husbandry but also in human populations.

  2. Plant Antifreeze Proteins and Their Expression Regulatory Mechanism

    Institute of Scientific and Technical Information of China (English)

    Lin Yuan-zhen; Lin Shan-zhi; Zhang Zhi-yi; Zhang Wei; Liu Wen-feng

    2005-01-01

    Low temperature is one of the major limiting environmental factors which constitutes the growth, development,productivity and distribution of plants. Over the past several years, the proteins and genes associated with freezing resistance of plants have been widely studied. The recent progress of domestic and foreign research on plant antifreeze proteins and the identification and characterization of plant antifreeze protein genes, especially on expression regulatory mechanism of plant antifreeze proteins are reviewed in this paper. Finally, some unsolved problems and the trend of research in physiological functions and gene expression regulatory mechanism of plant antifreeze proteins are discussed.

  3. Clinical characteristics and prognosis of acute myeloid leukemia associated with DNA-methylation regulatory gene mutations.

    Science.gov (United States)

    Ryotokuji, Takeshi; Yamaguchi, Hiroki; Ueki, Toshimitsu; Usuki, Kensuke; Kurosawa, Saiko; Kobayashi, Yutaka; Kawata, Eri; Tajika, Kenji; Gomi, Seiji; Kanda, Junya; Kobayashi, Anna; Omori, Ikuko; Marumo, Atsushi; Fujiwara, Yusuke; Yui, Shunsuke; Terada, Kazuki; Fukunaga, Keiko; Hirakawa, Tsuneaki; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Tamai, Hayato; Nakayama, Kazutaka; Wakita, Satoshi; Fukuda, Takahiro; Inokuchi, Koiti

    2016-09-01

    In recent years, it has been reported that the frequency of DNA-methylation regulatory gene mutations - mutations of the genes that regulate gene expression through DNA methylation - is high in acute myeloid leukemia. The objective of the present study was to elucidate the clinical characteristics and prognosis of acute myeloid leukemia with associated DNA-methylation regulatory gene mutation. We studied 308 patients with acute myeloid leukemia. DNA-methylation regulatory gene mutations were observed in 135 of the 308 cases (43.8%). Acute myeloid leukemia associated with a DNA-methylation regulatory gene mutation was more frequent in older patients (Pgene mutation was an unfavorable prognostic factor for overall survival in the whole cohort (P=0.0018), in patients aged ≤70 years, in patients with intermediate cytogenetic risk, and in FLT3-ITD-negative patients (P=0.0409). Among the patients with DNA-methylation regulatory gene mutations, 26.7% were found to have two or more such mutations and prognosis worsened with increasing number of mutations. In multivariate analysis DNA-methylation regulatory gene mutation was an independent unfavorable prognostic factor for overall survival (P=0.0424). However, patients with a DNA-methylation regulatory gene mutation who underwent allogeneic stem cell transplantation in first remission had a significantly better prognosis than those who did not undergo such transplantation (P=0.0254). Our study establishes that DNA-methylation regulatory gene mutation is an important unfavorable prognostic factor in acute myeloid leukemia.

  4. Modifier effects between regulatory and protein-coding variation.

    Directory of Open Access Journals (Sweden)

    Antigone S Dimas

    2008-10-01

    Full Text Available Genome-wide associations have shown a lot of promise in dissecting the genetics of complex traits in humans with single variants, yet a large fraction of the genetic effects is still unaccounted for. Analyzing genetic interactions between variants (epistasis is one of the potential ways forward. We investigated the abundance and functional impact of a specific type of epistasis, namely the interaction between regulatory and protein-coding variants. Using genotype and gene expression data from the 210 unrelated individuals of the original four HapMap populations, we have explored the combined effects of regulatory and protein-coding single nucleotide polymorphisms (SNPs. We predict that about 18% (1,502 out of 8,233 nsSNPs of protein-coding variants are differentially expressed among individuals and demonstrate that regulatory variants can modify the functional effect of a coding variant in cis. Furthermore, we show that such interactions in cis can affect the expression of downstream targets of the gene containing the protein-coding SNP. In this way, a cis interaction between regulatory and protein-coding variants has a trans impact on gene expression. Given the abundance of both types of variants in human populations, we propose that joint consideration of regulatory and protein-coding variants may reveal additional genetic effects underlying complex traits and disease and may shed light on causes of differential penetrance of known disease variants.

  5. Modifier effects between regulatory and protein-coding variation.

    Science.gov (United States)

    Dimas, Antigone S; Stranger, Barbara E; Beazley, Claude; Finn, Robert D; Ingle, Catherine E; Forrest, Matthew S; Ritchie, Matthew E; Deloukas, Panos; Tavaré, Simon; Dermitzakis, Emmanouil T

    2008-10-01

    Genome-wide associations have shown a lot of promise in dissecting the genetics of complex traits in humans with single variants, yet a large fraction of the genetic effects is still unaccounted for. Analyzing genetic interactions between variants (epistasis) is one of the potential ways forward. We investigated the abundance and functional impact of a specific type of epistasis, namely the interaction between regulatory and protein-coding variants. Using genotype and gene expression data from the 210 unrelated individuals of the original four HapMap populations, we have explored the combined effects of regulatory and protein-coding single nucleotide polymorphisms (SNPs). We predict that about 18% (1,502 out of 8,233 nsSNPs) of protein-coding variants are differentially expressed among individuals and demonstrate that regulatory variants can modify the functional effect of a coding variant in cis. Furthermore, we show that such interactions in cis can affect the expression of downstream targets of the gene containing the protein-coding SNP. In this way, a cis interaction between regulatory and protein-coding variants has a trans impact on gene expression. Given the abundance of both types of variants in human populations, we propose that joint consideration of regulatory and protein-coding variants may reveal additional genetic effects underlying complex traits and disease and may shed light on causes of differential penetrance of known disease variants.

  6. Functional Classification of Immune Regulatory Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Rubinstein, Rotem [Albert Einstein College of Medicine, Bronx, NY (United States); Ramagopal, Udupi A. [Albert Einstein College of Medicine, Bronx, NY (United States); Nathenson, Stanley G. [Albert Einstein College of Medicine, Bronx, NY (United States); Almo, Steven C. [Albert Einstein College of Medicine, Bronx, NY (United States); Fiser, Andras [Albert Einstein College of Medicine, Bronx, NY (United States)

    2013-05-01

    Members of the immunoglobulin superfamily (IgSF) control innate and adaptive immunity and are prime targets for the treatment of autoimmune diseases, infectious diseases, and malignancies. We describe a computational method, termed the Brotherhood algorithm, which utilizes intermediate sequence information to classify proteins into functionally related families. This approach identifies functional relationships within the IgSF and predicts additional receptor-ligand interactions. As a specific example, we examine the nectin/nectin-like family of cell adhesion and signaling proteins and propose receptor-ligand interactions within this family. We were guided by the Brotherhood approach and present the high-resolution structural characterization of a homophilic interaction involving the class-I MHC-restricted T-cell-associated molecule, which we now classify as a nectin-like family member. The Brotherhood algorithm is likely to have a significant impact on structural immunology by identifying those proteins and complexes for which structural characterization will be particularly informative.

  7. Sumoylation: a regulatory protein modification in health and disease.

    Science.gov (United States)

    Flotho, Annette; Melchior, Frauke

    2013-01-01

    Posttranslational modification with small ubiquitin-related modifier (SUMO) proteins is now established as one of the key regulatory protein modifications in eukaryotic cells. Hundreds of proteins involved in processes such as chromatin organization, transcription, DNA repair, macromolecular assembly, protein homeostasis, trafficking, and signal transduction are subject to reversible sumoylation. Hence, it is not surprising that disease links are beginning to emerge and that interference with sumoylation is being considered for intervention. Here, we summarize basic mechanisms and highlight recent developments in the physiology of sumoylation.

  8. CONSTRUCTION AND ANALYSIS OF IPBR/XYLS HYBRID REGULATORY PROTEINS

    Science.gov (United States)

    IpbR and XylS are related regulatory proteins (having 56% identity). IpbR responds to isopropylbenzene as well as to a variety of hydrophobic chemicals to activate expression of the isopropylbenzene catabolic pathway operon of pRE4 from ipbOP. XylS responds to substituted benzoic...

  9. Plant villins:Versatile actin regulatory proteins

    Institute of Scientific and Technical Information of China (English)

    Shanjin Huang; Xiaolu Qu; Ruihui Zhang

    2015-01-01

    Regulation of actin dynamics is a central theme in cel biology that is important for different aspects of cel physiology. Vil in, a member of the vil in/gelsolin/fragmin superfamily of proteins, is an important regulator of actin. Vil ins contain six gelsolin homology domains (G1–G6) and an extra headpiece domain. In contrast to their mammalian counterparts, plant vil ins are expressed widely, implying that plant vil ins play a more general role in regulating actin dynamics. Some plant vil ins have a defined role in modifying actin dynamics in the pol en tube;most of their in vivo activities remain to be ascertained. Recently, our understanding of the functions and mechanisms of action for plant vil ins has progressed rapidly, primarily due to the advent of Arabidopsis thaliana genetic approaches and imaging capabilities that can visualize actin dynamics at the single filament level in vitro and in living plant cel s. In this review, we focus on discussing the biochemical activities and modes of regulation of plant vil ins. Here, we present current understand-ing of the functions of plant vil ins. Final y, we highlight some of the key unanswered questions regarding the functions and regulation of plant vil ins for future research.

  10. Dynamical Analysis of Protein Regulatory Network in Budding Yeast Nucleus

    Institute of Scientific and Technical Information of China (English)

    LI Fang-Ting; JIA Xun

    2006-01-01

    @@ Recent progresses in the protein regulatory network of budding yeast Saccharomyces cerevisiae have provided a global picture of its protein network for further dynamical research. We simplify and modularize the protein regulatory networks in yeast nucleus, and study the dynamical properties of the core 37-node network by a Boolean network model, especially the evolution steps and final fixed points. Our simulation results show that the number of fixed points N(k) for a given size of the attraction basin k obeys a power-law distribution N(k)∝k-2.024. The yeast network is more similar to a scale-free network than a random network in the above dynamical properties.

  11. NF-kappaB regulatory mechanisms in alveolar macrophages from patients with acute respiratory distress syndrome.

    Science.gov (United States)

    Moine, P; McIntyre, R; Schwartz, M D; Kaneko, D; Shenkar, R; Le Tulzo, Y; Moore, E E; Abraham, E

    2000-02-01

    Activation of the nuclear regulatory factor NF-kappaB occurs in the lungs of patients with the acute respiratory distress syndrome (ARDS) and may contribute to the increased expression of immunoregulatory cytokines and other proinflammatory mediators in this setting. Because of the important role that NF-kappaB activation appears to play in the development of acute lung injury, we examined cytoplasmic and nuclear NF-kapppaB counterregulatory mechanisms, involving IkappaB proteins, in alveolar macrophages obtained from 7 control patients without lung injury and 11 patients with established ARDS. Cytoplasmic levels of the NF-kappaB subunits p50, p65, and c-Rel were significantly decreased in alveolar macrophages from patients with ARDS, consistent with enhanced migration of liberated NF-kappaB dimers from the cytoplasm to the nucleus. Cytoplasmic and nuclear levels of IkappaBalpha were not significantly altered in alveolar macrophages from patients with established ARDS, compared with controls. In contrast, nuclear levels of Bcl-3 were significantly decreased in patients with ARDS compared with controls (P = 0.02). No IkappaBgamma, IkappaBbeta, or p105 proteins were detected in the cytoplasm of alveolar macrophages from control patients or patients with ARDS. The presence of activated NF-kappaB in alveolar macrophages from patients with established ARDS implies the presence of an ongoing stimulus for NF-kappaB activation. In this setting, appropriate counterregulatory mechanisms to normalize nuclear levels of NF-kappaB and to suppress NF-kappaB-mediated transcription, such as increased cytoplasmic and nuclear IkappaBalpha levels or decreased Bcl-3 levels, appeared to be induced. Nevertheless, even though counterregulatory mechanisms to NF-kappaB activation are activated in lung macrophages of patients with ARDS, NF-kappaB remains activated. These results suggest that fundamental abnormalities in transcriptional mechanisms involving NF-kappaB and important in the

  12. Promoters, Transcripts, and Regulatory Proteins of Mungbean Yellow Mosaic Geminivirus†

    OpenAIRE

    Shivaprasad, P. V.; Akbergenov, Rashid; Trinks, Daniela; R Rajeswaran; Veluthambi, K; Hohn, Thomas; Pooggin, Mikhail M.

    2005-01-01

    Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virus-Vigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and ...

  13. Crystal structure of nitrogen regulatory protein IIANtr from Neisseria meningitidis

    Directory of Open Access Journals (Sweden)

    Stammers David K

    2005-08-01

    Full Text Available Abstract Background The NMB0736 gene of Neisseria meningitidis serogroup B strain MC58 encodes the putative nitrogen regulatory protein, IIANtr (abbreviated to NM-IIANtr. The homologous protein present in Escherichia coli is implicated in the control of nitrogen assimilation. As part of a structural proteomics approach to the study of pathogenic Neisseria spp., we have selected this protein for structure determination by X-ray crystallography. Results The NM-IIANtr was over-expressed in E. coli and was shown to be partially mono-phosphorylated, as assessed by mass spectrometry of the purified protein. Crystals of un-phosphorylated protein were obtained and diffraction data collected to 2.5 Å resolution. The structure of NM-IIANtr was solved by molecular replacement using the coordinates of the E. coli nitrogen regulatory protein IIAntr [PDB: 1A6J] as the starting model. The overall fold of the Neisseria enzyme shows a high degree of similarity to the IIANtr from E. coli, and the position of the phosphoryl acceptor histidine residue (H67 is conserved. The orientation of an adjacent arginine residue (R69 suggests that it may also be involved in coordinating the phosphate group. Comparison of the structure with that of E. coli IIAmtl complexed with HPr [PDB: 1J6T] indicates that NM-IIANtr binds in a similar way to the HPr-like enzyme in Neisseria. Conclusion The structure of NM-IIANtr confirms its assignment as a homologue of the IIANtr proteins found in a range of other Gram-negative bacteria. We conclude that the NM- IIANtr protein functions as part of a phosphorylation cascade which, in contrast to E. coli, shares the upstream phosphotransfer protein with the sugar uptake phosphoenolpyruvate:sugar phosphotransferase system (PTS, but in common with E. coli has a distinct downstream effector mechanism.

  14. Laboratory tests for disorders of complement and complement regulatory proteins.

    Science.gov (United States)

    Shih, Angela R; Murali, Mandakolathur R

    2015-12-01

    The complement pathway is a cascade of proteases that is involved in immune surveillance and innate immunity, as well as adaptive immunity. Dysfunction of the complement cascade may be mediated by aberrations in the pathways of activation, complement regulatory proteins, or complement deficiencies, and has been linked to a number of hematologic disorders, including paroxysmal noctural hemoglobinuria (PNH), hereditary angioedema (HAE), and atypical hemolytic-uremic syndrome (aHUS). Here, current laboratory tests for disorders of the complement pathway are reviewed, and their utility and limitations in hematologic disorders and systemic diseases are discussed. Current therapeutic advances targeting the complement pathway in treatment of complement-mediated hematologic disorders are also reviewed.

  15. Exploitation of complement regulatory proteins by Borrelia and Francisella.

    Science.gov (United States)

    Madar, Marian; Bencurova, Elena; Mlynarcik, Patrik; Almeida, André M; Soares, Renata; Bhide, Katarina; Pulzova, Lucia; Kovac, Andrej; Coelho, Ana V; Bhide, Mangesh

    2015-06-01

    Pathogens have developed sophisticated mechanisms of complement evasion such as binding to the host complement regulatory proteins (CRPs) on their surface or expression of CRP mimicking molecules. The ability of pathogens to evade the complement system has been correlated with pathogenesis and host selectivity. Hitherto, little work has been undertaken to determine whether Borrelia and Francisella exploit various CRPs to block complement attack. Seventeen Borrelia (twelve species) and six Francisella (three subspecies) strains were used to assess their ability to bind human, sheep and cattle CRPs or mimic membrane associated complement regulators. A series of experiments including affinity ligand binding experiments, pull-down assays and mass spectrometry based protein identification, revealed an array of CRP binding proteins of Borrelia and Francisella. Unlike Francisella, Borrelia strains were able to bind multiple human CRPs. Three strains of Borrelia (SKT-4, SKT-2 and HO14) showed the presence of a human CD46-homologous motif, indicating their ability to possess putative human CD46 mimicking molecules. Similarly, five strains of Borrelia and two strains of Francisella may have surface proteins with human CD59-homologous motifs. Among ovine and bovine CRPs, the only CRP bound by Francisella (LVS, Tul4 strain) was vitronectin, while ovine C4BP, ovine factor H and bovine factor H were bound to Borrelia strains SKT-2, DN127 and Co53. This study presents an array of proteins of Borrelia and Francisella that bind CRPs or may mimic membrane-CRPs, thus enabling multiphasic complement evasion strategies of these pathogens.

  16. Promoters, Transcripts, and Regulatory Proteins of Mungbean Yellow Mosaic Geminivirus†

    Science.gov (United States)

    Shivaprasad, P. V.; Akbergenov, Rashid; Trinks, Daniela; Rajeswaran, R.; Veluthambi, K.; Hohn, Thomas; Pooggin, Mikhail M.

    2005-01-01

    Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virus-Vigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing. PMID:15956560

  17. Promoters, transcripts, and regulatory proteins of Mungbean yellow mosaic geminivirus.

    Science.gov (United States)

    Shivaprasad, P V; Akbergenov, Rashid; Trinks, Daniela; Rajeswaran, R; Veluthambi, K; Hohn, Thomas; Pooggin, Mikhail M

    2005-07-01

    Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virus-Vigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing.

  18. Iron Regulatory Proteins Mediate Host Resistance to Salmonella Infection.

    Science.gov (United States)

    Nairz, Manfred; Ferring-Appel, Dunja; Casarrubea, Daniela; Sonnweber, Thomas; Viatte, Lydie; Schroll, Andrea; Haschka, David; Fang, Ferric C; Hentze, Matthias W; Weiss, Guenter; Galy, Bruno

    2015-08-12

    Macrophages are essential for systemic iron recycling, and also control iron availability to pathogens. Iron metabolism in mammalian cells is orchestrated posttranscriptionally by iron-regulatory proteins (IRP)-1 and -2. Here, we generated mice with selective and combined ablation of both IRPs in macrophages to investigate the role of IRPs in controlling iron availability. These animals are hyperferritinemic but otherwise display normal clinical iron parameters. However, mutant mice rapidly succumb to systemic infection with Salmonella Typhimurium, a pathogenic bacterium that multiplies within macrophages, with increased bacterial burdens in liver and spleen. Ex vivo infection experiments indicate that IRP function restricts bacterial access to iron via the EntC and Feo bacterial iron-acquisition systems. Further, IRPs contain Salmonella by promoting the induction of lipocalin 2, a host antimicrobial factor that inhibits bacterial uptake of iron-laden siderophores, and by suppressing the ferritin iron pool. This work reveals the importance of the IRPs in innate immunity.

  19. Enzymatic Mercury Detoxification: The Regulatory Protein MerR

    CERN Multimedia

    Ctortecka, B; Walsh, C T; Comess, K M

    2002-01-01

    Mercury ions and organomercurial reagents are extremely toxic due to their affinity for thiol groups. Many bacteria contain an elaborate detoxification system for a metabolic conversion of toxic Hg$^{2+}$ or organomercurials to less toxic elemental Hg$^0$. The main components of the enzymatic mercury detoxification (see Fig. 1) are the regulatory protein MerR (mercury responsive genetic switch), the organomercurial lyase MerB (cleavage of carbon mercury bonds), and the mercuric ion reductase MerA (reduction of mercuric ions). In these proteins Hg$^{2+}$ is usually coordinated by the thiol groups of cysteines. We utilize the nuclear quadrupole interaction (NQI) of ${\\rm^{199m}}$Hg detected by time differential perturbed angular correlation (TDPAC) to identify the Hg metal site geometries in these proteins in order to elucidate the molecular origin of the ultrasensitivity, selectivity and reaction mechanism of this detoxification system. The short lived TDPAC probe ${\\rm^{199m}}$Hg ($\\tau_{1/2} =$ 43 min) is su...

  20. Impaired circulating CD4+ LAP+ regulatory T cells in patients with acute coronary syndrome and its mechanistic study.

    Directory of Open Access Journals (Sweden)

    Zheng-Feng Zhu

    Full Text Available OBJECTIVE: CD4(+ latency-associated peptide (LAP(+ regulatory T cells (Tregs are a newly discovered T cell subset in humans and the role of these cells in patients with acute coronary syndrome (ACS has not been explored. We designed to investigate whether circulating frequency and function of CD4(+LAP(+ Tregs are defective in ACS. METHODS: One hundred eleven ACS patients (acute myocardial infarction and unstable angina and 117 control patients were enrolled in the study. The control patients consisted of chronic stable angina (CSA and chest pain syndrome (CPS. The frequencies of circulating CD4(+LAP(+ Tregs and the expression of the transmembrane protein glycoprotein-A repetitions predominant (GARP on CD4(+ T cells were determined by flow cytometry. The function of CD4(+LAP(+ Tregs was detected using thymidine uptake. Serum interleukin-10 (IL-10 and transforming growth factor-β protein (TGF-β levels were detected using ELISA and expression of GARP mRNA in peripheral blood mononuclear cells (PBMCs was measured by real time-polymerase chain reaction. RESULTS: We found ACS patients had a significantly lower frequency of circulating CD4(+LAP(+ Tregs, and the function of these cells was reduced compared to controls. The expression of GARP in CD4(+ T cells and the serum levels of TGF-β in ACS patients were lower than those of control patients. The serum levels of IL-10 were similar between the two cohorts. CONCLUSIONS: A novel regulatory T cell subset, defined as CD4(+LAP(+ T cells is defective in ACS patients.

  1. Maintaining cholesterol homeostasis:Sterol regulatory element-binding proteins

    Institute of Scientific and Technical Information of China (English)

    Lutz W. Weber; Meinrad Boll; Andreas Stampfl

    2004-01-01

    The molecular mechanism of how hepatocytes maintain cholesterol homeostasis has become much more transparent with the discovery of sterol regulatory element binding proteins (SREBPs) in recent years. These membrane proteins are members of the basic helix-loop-helix-leucine zipper (bHLHZip) family of transcription factors. They activate the expression of at least 30 genes involved in the synthesis of cholesterol and lipids. SREBPs are synthesized as precursor proteins in the endoplasmic reticulum (ER), where they form a complex with another protein, SREBP cleavage activating protein (SCAP).The SCAP molecule contains a sterol sensory domain. In the presence of high cellular sterol concentrations SCAP confines SREBP to the ER. With low cellular concentrations, SCAP escorts SREBP to activation in the Golgi. There, SREBP undergoes two proteolytic cleavage steps to release the mature, biologically active transcription factor, nuclear SREBP (nSREBP). nSREBP translocates to the nucleus and binds to sterol response elements (SRE) in the promoter/enhancer regions of target genes. Additional transcription factors are required to activate transcription of these genes. Three different SREBPs are known, SREBPs-1a, -1c and -2. SREBP-1a and -1c are isoforms produced from a single gene by alternate splicing. SREBP-2is encoded by a different gene and does not display any isoforms. It appears that SREBPs alone, in the sequence described above, can exert complete control over cholesterol synthesis, whereas many additional factors (hormones,cytokines, etc.) are required for complete control of lipid metabolism. Medicinal manipulation of the SREBP/SCAP system is expected to prove highly beneficial in the management of cholesterol-related disease.

  2. Pleiotropy constrains the evolution of protein but not regulatory sequences in a transcription regulatory network influencing complex social behaviours

    Directory of Open Access Journals (Sweden)

    Daria eMolodtsova

    2014-12-01

    Full Text Available It is increasingly apparent that genes and networks that influence complex behaviour are evolutionary conserved, which is paradoxical considering that behaviour is labile over evolutionary timescales. How does adaptive change in behaviour arise if behaviour is controlled by conserved, pleiotropic, and likely evolutionary constrained genes? Pleiotropy and connectedness are known to constrain the general rate of protein evolution, prompting some to suggest that the evolution of complex traits, including behaviour, is fuelled by regulatory sequence evolution. However, we seldom have data on the strength of selection on mutations in coding and regulatory sequences, and this hinders our ability to study how pleiotropy influences coding and regulatory sequence evolution. Here we use population genomics to estimate the strength of selection on coding and regulatory mutations for a transcriptional regulatory network that influences complex behaviour of honey bees. We found that replacement mutations in highly connected transcription factors and target genes experience significantly stronger negative selection relative to weakly connected transcription factors and targets. Adaptively evolving proteins were significantly more likely to reside at the periphery of the regulatory network, while proteins with signs of negative selection were near the core of the network. Interestingly, connectedness and network structure had minimal influence on the strength of selection on putative regulatory sequences for both transcription factors and their targets. Our study indicates that adaptive evolution of complex behaviour can arise because of positive selection on protein-coding mutations in peripheral genes, and on regulatory sequence mutations in both transcription factors and their targets throughout the network.

  3. Protein modularity, cooperative binding, and hybrid regulatory states underlie transcriptional network diversification.

    Science.gov (United States)

    Baker, Christopher R; Booth, Lauren N; Sorrells, Trevor R; Johnson, Alexander D

    2012-09-28

    We examine how different transcriptional network structures can evolve from an ancestral network. By characterizing how the ancestral mode of gene regulation for genes specific to a-type cells in yeast species evolved from an activating paradigm to a repressing one, we show that regulatory protein modularity, conversion of one cis-regulatory sequence to another, distribution of binding energy among protein-protein and protein-DNA interactions, and exploitation of ancestral network features all contribute to the evolution of a novel regulatory mode. The formation of this derived mode of regulation did not disrupt the ancestral mode and thereby created a hybrid regulatory state where both means of transcription regulation (ancestral and derived) contribute to the conserved expression pattern of the network. Finally, we show how this hybrid regulatory state has resolved in different ways in different lineages to generate the diversity of regulatory network structures observed in modern species.

  4. Acute phase proteins as diagnostic markers in horses with colic

    DEFF Research Database (Denmark)

    Pihl, Tina; Scheepers, Elrien; Sanz, Macarena;

    2016-01-01

    Objective – To investigate the diagnostic potential of the concentrations of acute-phase proteins serum amyloid A (SAA), haptoglobin (Hp), and fibrinogen in blood and peritoneal fluid (PF) for differentiating horses within flammatory colic (entero-colitis and peritonitis) from those with surgical...... colic. Design – Prospective observational multicenter study. Setting – Two university referral hospitals. Animals – Horses referred for severe acute abdominal pain to Hospital 1 (n = 148) or Hospital 2 (n = 78). Intervention – Blood and PF samples collected at admission were used for acute-phase protein...... concentrations) did not improve the model. When validated in Hospital 2 data, the models had good integrity and diagnostic performance. Conclusions – Evaluation of SAA in serum improved the ability to differentiate horses with acute inflammatory colic requiring medical treatment from horses with colic requiring...

  5. Protein C and S deficiency presenting as acute abdomen

    Directory of Open Access Journals (Sweden)

    Amit A Bharadiya

    2015-01-01

    Full Text Available Protein C and S are essential in limiting the activation of coagulation in vivo. Their deficiencies predispose the patient to thrombophilia and leads to thrombosis, often at unusual sites. Arterial thrombosis is rarely observed. We report a case of a patient with abdominal arteriovenous thrombosis leading to multiorgan infarction secondary to deficiency of protein C and protein S and presenting as acute abdomen.

  6. The Evolution of the Secreted Regulatory Protein Progranulin.

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    Roger G E Palfree

    Full Text Available Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i: the origins of metazoan progranulins (ii: the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii: the evolution of granulin module architectures of vertebrate progranulins (iv: the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl

  7. 不同月龄和血脂水平ApoE基因敲除小鼠体内类固醇激素合成急性调节蛋白的表达%Expression of steroidogenic acute regulatory protein following with age and serum lipids levels in apolipoprotein E-deficient mice

    Institute of Scientific and Technical Information of China (English)

    宁艳霞; 江一峰; 徐晨; 赵凤娣; 殷莲华

    2010-01-01

    目的 检测不同月龄和不同血脂水平载脂蛋白E敲除小鼠体内类固醇激素合成急性调节蛋白(steroidogenic acute regulatory protein,StAR)的表达水平.方法 实验分别选取雄性和雌性1天龄新生鼠、1月龄、3月龄及5月龄载脂蛋白E敲除小鼠(apoE-/-)及对照C57BL/6J小鼠,每组6只,共16组.利用试剂盒检测不同年龄小鼠的血脂水平.利用实时定量RT-PCR及Western blot方法检测不同年龄和血脂水平下,小鼠肝脏中StAR基因和蛋白的表达.结果 相比同年龄同性别的正常对照小鼠,载脂蛋白E敲除小鼠血清中含较高水平的低密度脂蛋白(LDL)和较低水平的高密度脂蛋白(HDL).在对照小鼠中,StAR基因和蛋白表达水平随月龄增加逐渐下降;但是在载脂蛋白E敲除小鼠体内,因为血脂水平的提高,StAR基因和蛋白表达水平呈现先增高后降低的趋势.结论 StAR通过调节脂质代谢,可作为一个有效调节动脉粥样硬化以及其他心血管疾病的调节因子.

  8. Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors

    Science.gov (United States)

    Lloyd, David J.; St Jean, David J.; Kurzeja, Robert J. M.; Wahl, Robert C.; Michelsen, Klaus; Cupples, Rod; Chen, Michelle; Wu, John; Sivits, Glenn; Helmering, Joan; Komorowski, Renée; Ashton, Kate S.; Pennington, Lewis D.; Fotsch, Christopher; Vazir, Mukta; Chen, Kui; Chmait, Samer; Zhang, Jiandong; Liu, Longbin; Norman, Mark H.; Andrews, Kristin L.; Bartberger, Michael D.; van, Gwyneth; Galbreath, Elizabeth J.; Vonderfecht, Steven L.; Wang, Minghan; Jordan, Steven R.; Véniant, Murielle M.; Hale, Clarence

    2013-12-01

    Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

  9. Activation of counter-regulatory mechanisms in a rat renal acute rejection model

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    Salomon Daniel R

    2008-02-01

    Full Text Available Abstract Background Microarray analysis provides a powerful approach to identify gene expression alterations following transplantation. In patients the heterogeneity of graft specimens, co-morbidity, co-medications and the challenges in sample collection and preparation complicate conclusions regarding the underlying mechanisms of graft injury, rejection and immune regulation. Results We used a rat kidney transplantation model with strict transplant and sample preparation procedures to analyze genome wide changes in gene expression four days after syngeneic and allogeneic transplantation. Both interventions were associated with substantial changes in gene expression. After allogeneic transplantation, genes and pathways related to transport and metabolism were predominantly down-regulated consistent with rejection-mediated graft injury and dysfunction. Up-regulated genes were primarily related to the acute immune response including antigen presentation, T-cell receptor signaling, apoptosis, interferon signaling and complement cascades. We observed a cytokine and chemokine expression profile consistent with activation of a Th1-cell response. A novel finding was up-regulation of several regulatory and protective genes after allogeneic transplantation, specifically IL10, Bcl2a1, C4bpa, Ctla4, HO-1 and the SOCS family. Conclusion Our data indicate that in parallel with the predicted activation of immune response and tissue injury pathways, there is simultaneous activation of pathways for counter regulatory and protective mechanisms that would balance and limit the ongoing inflammatory/immune responses. The pathophysiological mechanisms behind and the clinical consequences of alterations in expression of these gene classes in acute rejection, injury and dysfunction vs. protection and immunoregulation, prompt further analyses and open new aspects for therapeutic approaches.

  10. Evaluation of Regulatory T Cells in Patients with Acute and Chronic Brucellosis

    Science.gov (United States)

    Ganji, Ali; Mosayebi, Ghasem; Ghaznavi-Rad, Ehsanollah; khosravi, Khadije; Zarinfar, Nader

    2017-01-01

    Background: Brucellosis is one of the most common chronic diseases, with widespread distribution. In spite of cell-mediated immunity (CMI) modulated mainly via activated T-helper type 1 (Th1) cells, brucellosis can advance to chronic disease in about 10-30% of cases. Regulatory T cells (Treg cells) are involved the immune response to brucellosis; however, their role, particularly in the change from the acute to the chronic phase, have not yet been elucidated. The main hypothesis of this study was that Treg cells play critical roles in the progression of brucellosis from the acute to the chronic phase. Methods: Forty-eight unrelated subjects participated in this case-control study. The percentages of CD4+, CD25+, FoxP3+, and CD25/FoxP3+ T cells in the peripheral blood mononuclear cells (PBMCs) of acute (AB) and chronic brucellosis (CB) patients and healthy controls were determined by flow cytometry. The mean florescence intensities (MFIs) of CD4+, CD25+, and FoxP3+ T cells were also measured. Results: We found a significantly lower percentage of CD25/FoxP3+ Treg cells in CB than in the AB and control groups (p < 0.05). Also, CD4 and CD25 MFIs were significantly less in CB than in AB and controls (p < 0.05). Conclusions: We propose that the reduced number of CD25/FoxP3+ Treg cells in the CB group leads to T cell anergy and this contributes to the development of chronic infection. PMID:28367469

  11. Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling

    Science.gov (United States)

    Zhao, Yang; Xie, Shaojun; Batelli, Giorgia; Wang, Bangshing; Duan, Cheng-Guo; Wang, Xingang; Xing, Lu; Lei, Mingguang; Yan, Jun; Zhu, Xiaohong; Zhu, Jian-Kang

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling. PMID:26943172

  12. Application of acute phase protein measurements in veterinary clinical chemistry

    DEFF Research Database (Denmark)

    Petersen, Henning; Nielsen, J. P.; Heegaard, Peter M. H.

    2004-01-01

    The body's early defence in response to trauma, inflammation or infection, the acute phase response, is a complex set of systemic reactions seen shortly after exposure to a triggering event. One of the many components is an acute phase protein response in which increased hepatic synthesis leads t...... A and their possible use as non-specific indicators of health in large animal veterinary medicine such as in the health status surveillance of pigs at the herd level, for the detection of mastitis in dairy cattle and for the prognosis of respiratory diseases in horses....

  13. Redox Modulation of Cellular Signaling and Metabolism Through Reversible Oxidation of Methionine Sensors in Calcium Regulatory Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bigelow, Diana J.; Squier, Thomas C.

    2005-01-17

    Adaptive responses associated with environmental stressors are critical to cell survival. These involve the modulation of central signaling protein functions through site-specific and enzymatically reversible oxidative modifications of methionines to coordinate cellular metabolism, energy utilization, and calcium signaling. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. Thus, depending on either the ecological niche of the organism or the cellular milieu of different organ systems, cellular metabolism can be fine-tuned to maintain optimal function in the face of variable amounts of collateral oxidative damage. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met{sup 144} or Met{sup 145} results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylationdependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in

  14. Development of coagulation regulatory proteins in the fetal and neonatal lamb.

    Science.gov (United States)

    Manco-Johnson, Marilyn J; Jacobson, Linda J; Hacker, Michele R; Townsend, Susan F; Murphy, James; Hay, William

    2002-10-01

    To investigate the development of coagulation regulatory proteins-protein C (PC), protein S (PS), and antithrombin (AT)-in relationship to the procoagulant protein factor X (FX), a chronically catheterized fetal ovine model was used. Infusion and sampling catheters were placed into pregnant ewes and their fetuses and maintained from mid-gestation. From a total of 110 fetuses, 17 lambs, and 63 ewes that were studied on one to 15 occasions, 212 fetal, 88 neonatal, and 157 maternal samples were obtained. Liver tissue was obtained from 31 fetuses and 15 ewes. Plasma levels of all proteins studied were higher in the ewe than in the fetus (p 0.05). This study suggests that fetal regulation of coagulation proteins follows characteristic patterns relative to the vitamin K dependence of the protein rather than its role as a procoagulant versus regulatory protein.

  15. Alterations of proteins in MDCK cells during acute potassium deficiency.

    Science.gov (United States)

    Peerapen, Paleerath; Ausakunpipat, Nardtaya; Chanchaem, Prangwalai; Thongboonkerd, Visith

    2016-06-01

    Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury.

  16. Shotgun Proteomics Identifies Proteins Specific for Acute Renal Transplant Rejection

    Science.gov (United States)

    Sigdel, Tara K.; Kaushal, Amit; Gritsenko, Marina; Norbeck, Angela D.; Qian, Wei-Jun; Xiao, Wenzhong; Camp, David G.; Smith, Richard D.; Sarwal, Minnie M.

    2010-01-01

    Acute rejection (AR) remains the primary risk factor for renal transplant outcome; development of non-invasive diagnostic biomarkers for AR is an unmet need. We used shotgun proteomics applying LC-MS/MS and ELISA to analyze a set of 92urine samples, from patients with AR, stable grafts (STA), proteinuria (NS), and healthy controls (HC). A total of 1446 urinary proteins were identified along with a number of NS specific, renal transplantation specific and AR specific proteins. Relative abundance of identified urinary proteins was measured by protein-level spectral counts adopting a weighted fold-change statistic, assigning increased weight for more frequently observed proteins. We have identified alterations in a number of specific urinary proteins in AR, primarily relating to MHC antigens, the complement cascade and extra-cellular matrix proteins. A subset of proteins (UMOD, SERPINF1 and CD44), have been further cross-validated by ELISA in an independent set of urine samples, for significant differences in the abundance of these urinary proteins in AR. This label-free, semi-quantitative approach for sampling the urinary proteome in normal and disease states provides a robust and sensitive method for detection of urinary proteins for serial, non-invasive clinical monitoring for graft rejection after kidney transplantation. PMID:20543976

  17. Shotgun Proteomics Identifies Proteins Specific for Acute Renal Transplant Rejection

    Energy Technology Data Exchange (ETDEWEB)

    Sigdel, Tara K.; Kaushal, Amit; Gritsenko, Marina A.; Norbeck, Angela D.; Qian, Weijun; Xiao, Wenzhong; Camp, David G.; Smith, Richard D.; Sarwal, Minnie M.

    2010-01-04

    Acute rejection (AR) remains the primary risk factor for renal transplant outcome; development of non-invasive diagnostic biomarkers for AR is an unmet need. We used shotgun proteomics using LC-MS/MS and ELISA to analyze a set of 92 urine samples, from patients with AR, stable grafts (STA), proteinuria (NS), and healthy controls (HC). A total of 1446 urinary proteins were identified along with a number of NS specific, renal transplantation specific and AR specific proteins. Relative abundance of identified urinary proteins was measured by protein-level spectral counts adopting a weighted fold-change statistic, assigning increased weight for more frequently observed proteins. We have identified alterations in a number of specific urinary proteins in AR, primarily relating to MHC antigens, the complement cascade and extra-cellular matrix proteins. A subset of proteins (UMOD, SERPINF1 and CD44), have been further cross-validated by ELISA in an independent set of urine samples, for significant differences in the abundance of these urinary proteins in AR. This label-free, semi-quantitative approach for sampling the urinary proteome in normal and disease states provides a robust and sensitive method for detection of urinary proteins for serial, non-invasive clinical monitoring for graft rejection after

  18. Analysis of protein phosphatase-1 and aurora protein kinase suppressors reveals new aspects of regulatory protein function in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Anuprita Ghosh

    Full Text Available Protein phosphatase-1 (PP1 controls many processes in eukaryotic cells. Modulation of mitosis by reversing phosphorylation of proteins phosphorylated by aurora protein kinase is a critical function for PP1. Overexpression of the sole PP1, Glc7, in budding yeast, Saccharomyces cerevisiae, is lethal. This work shows that lethality requires the function of Glc7 regulatory proteins Sds22, Reg2, and phosphorylated Glc8. This finding shows that Glc7 overexpression induced cell death requires a specific subset of the many Glc7-interacting proteins and therefore is likely caused by promiscuous dephosphorylation of a variety of substrates. Additionally, suppression can occur by reducing Glc7 protein levels by high-copy Fpr3 without use of its proline isomerase domain. This divulges a novel function of Fpr3. Most suppressors of GLC7 overexpression also suppress aurora protein kinase, ipl1, temperature-sensitive mutations. However, high-copy mutant SDS22 genes show reciprocal suppression of GLC7 overexpression induced cell death or ipl1 temperature sensitivity. Sds22 binds to many proteins besides Glc7. The N-terminal 25 residues of Sds22 are sufficient to bind, directly or indirectly, to seven proteins studied here including the spindle assembly checkpoint protein, Bub3. These data demonstrate that Sds22 organizes several proteins in addition to Glc7 to perform functions that counteract Ipl1 activity or lead to hyper Glc7 induced cell death. These data also emphasize that Sds22 targets Glc7 to nuclear locations distinct from Ipl1 substrates.

  19. No relationship between most polymorphisms of steroidogenic acute regulatory (StAR gene with polycystic ovarian syndrome

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    Azadeh-Sadat Nazouri

    2015-12-01

    Full Text Available Background: Polycystic ovary syndrome (PCOS is one of the most common endocrine women’s disorders in reproductive age. Hyperandrogenism has a critical role in the etiology of PCOS and it can cause fault in Steroidogenesis process. During steroidogenesis, steroidogenic acute regulatory protein (StAR seems to increase the delivery of cholesterol through mitochondrial membrane. Therefore, polymorphisms of StAR might effect on this protein and play a role in the etiology of PCOS. Objective: The aim of this study was to investigate the association between StAR SNPs with PCOS. Thus, seven polymorphisms in this gene: rs104894086, rs104894089, rs104894090, rs137852689, rs10489487, rs104894085 were detected. Materials and Methods: In this case control study, 45 PCOS women, 40 male factor/unexplained infertile women, and 40 fertile women as two control groups were participated from 2008-2012. Polymorphisms were detected using restriction fragment length polymorphism (PCR-RFLP method. Results: Heterozygote genotyping for rs137852689 SNP (amino acid 218 C > T was only seen in seven PCOS patients, one in normal ovulatory women, and five in male factor/unexplained infertile women (15.5%, 2.5%, 12.5%, respectively (p= 0.12. While, it has shown no association between other SNPS with PCOs. Conclusion: The RFLP results for seven chosen SNPs, which located in exon 5 and 7 showed normal status in three groups, it means no heterozygous or homozygous forms of selected SNPs were observed. So, it seems evaluation of the active amino acid sites should be investigated and also the study population should be increased.

  20. Acute phase protein response in the capybara (Hydrochoerus hydrochaeris).

    Science.gov (United States)

    Bernal, Luis; Feser, Mariane; Martínez-Subiela, Silvia; García-Martínez, Juan D; Cerón, José J; Tecles, Fernando

    2011-10-01

    We evaluated the acute phase protein response in capybaras (Hydrochoerus hydrochaeris). Three animal groups were used: 1) healthy animals (n=30), 2) a group in which experimental inflammation with turpentine was induced (n=6), and 3) a group affected with sarcoptic scabies (n=14) in which 10 animals were treated with ivermectin. Haptoglobin (Hp), acid-soluble glycoprotein (ASG) and albumin were analyzed in all animals. In those treated with turpentine, Hp reached its maximum value at 2 wk with a 2.7-fold increase, whereas ASG increased 1.75-fold and albumin decreased 0.87-fold 1 wk after the induction of inflammation. Capybaras affected with sarcoptic scabies presented increases in Hp and ASG of 4.98- and 3.18-fold, respectively, and a 0.87-fold decrease in albumin, compared with healthy animals. Haptoglobin and ASG can be considered as moderate, positive acute phase proteins in capybaras because they showed less than 10-fold increases after an inflammatory process and reached their peak concentrations 1 wk after the induction of inflammation. Conversely, albumin can be considered a negative acute phase protein in capybaras because it showed a reduction in concentration after inflammatory stimulus.

  1. Acute HIV Seroconversion Presenting with Active Tuberculosis and Associated with High Levels of T-Regulatory Cells

    Science.gov (United States)

    Sued, Omar; Quiroga, María Florencia; Socías, María Eugenia; Turk, Gabriela; Salomón, Horacio

    2011-01-01

    Abstract A patient with well-defined acute HIV infection who developed concomitant pulmonary tuberculosis during the retroviral acute syndrome is reported here. In this patient high levels of T-regulatory cells (Tregs) and a low proliferation response to M. tuberculosis were initially detected, which normalized throughout follow-up. This case calls for the consideration of tuberculosis in patients in the early stages of HIV, and emphasizes the need for further study of the potential causal relationship between Treg cells and the risk of TB reactivation in HIV patients. PMID:21774688

  2. Microheterogeneity of acute phase proteins in patients with ulcerative colitis

    Institute of Scientific and Technical Information of China (English)

    Marian Grzymis(l)awski; Katarzyna Derc; Magdalena Sobieska; Krzysztof Wiktorowicz

    2006-01-01

    AIM: To estimate the serum α1-antichymotrypsin (ACT),α1-acid glycoprotein (AGP) and transferrin (Tf) concentrations and to evaluate the microheterogeneity of these acute phase proteins in patients with ulcerative colitis. METHODS: Twenty-seven patients with ulcerative colitis (UC) and 17 healthy control subjects were studied. The patients were categorised as severe (n = 9), moderate (n = 10) and mild groups (n = 8) using Truelove and Witts'classification of ulcerative colitis. Microheterogeneity of ACT, AGP and Tf was analysed by crossed immunoaffinity electrophoresis (CIAE) with concanavalin A. In all serum samples standard electrophoresis of serum proteins was performed, iron (Fe) concentration, total iron binding capacity (TIBC) and C-reactive protein (CRP) were also measured.RESULTS: Our patients suffering from ulcerative colitis had significantly higher serum ACT and AGP concentrations and lower serum transferrin concentration in comparison to healthy subjects. Changes in concentrationsof acute phase proteins were dependent on the activityof the inflammatory process. The glycosylation patterns of transferrin were related to the inflammation status. We also observed the correlation between ACT and AGP concentrations, patterns of transferrin glycosylation and changes in standard protein electrophoresis or blood cell count.CONCLUSION: The glycosylation patterns of transferrin obtained from patients suffering from ulcerative colitis are highly branched and sialylated compared with those obtained from healthy subjects. In contrast, the glycosylation patterns of transferrin do not differ according to the activity index of ulcerative colitis. The microheterogeneity patterns of AGP and ACT are similar in ulcerative colitis patients and healthy subjects.

  3. Regulatory T cells-derived IL-35 promotes the growth of adult acute myeloid leukemia blasts.

    Science.gov (United States)

    Tao, Qianshan; Pan, Ying; Wang, Yiping; Wang, Huiping; Xiong, Shudao; Li, Qing; Wang, Jia; Tao, Lili; Wang, Zhitao; Wu, Fan; Zhang, Rui; Zhai, Zhimin

    2015-11-15

    Tumor immune escape mechanism mediated by CD4+CD25+regulatory T cells (Tregs) is a key factor in the pathogenesis of acute myeloid leukemia (AML). IL-35, as a novel inhibitory cytokine, is produced by Tregs specially and regulates functions of Tregs in murine. However, IL-35 expression of Tregs in human is still disputed, and its role in AML is yet to be elucidated. In this study, we found that IL-35 was expressed highly in peripheral blood plasma of adult patients with AML and significantly correlated with the clinical stages of malignancy. Tregs-derived from adult AML patients produced IL-35 in a stimulation-dependent manner. IL-35 promoted AML blasts immune escape by expanding Tregs and inhibiting CD4+CD25-effector T cells (Teffs). Furthermore, IL-35 directly promoted the proliferation of AML blasts and reduced the apoptosis of AML blasts. Together, our study demonstrates that IL-35-derived from Tregs promotes the growth of adult AML blasts, suggesting that IL-35 has an important role in the pathogenesis of AML.

  4. Therapeutic reactivation of protein phosphatase 2A in acute myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Kavitha eRamaswamy

    2015-02-01

    Full Text Available Protein phosphatase 2A (PP2A is a serine/threonine phosphatase that is required for normal cell growth and development. PP2A is a potent tumor suppressor, which is inactivated in cancer cells as a result of genetic deletions and mutations. In myeloid leukemias, genes encoding PP2A subunits are generally intact. Instead, PP2A is functionally inhibited by post-translational modifications of its catalytic C subunit, and interactions with negative regulators by its regulatory B and scaffold A subunits. Here, we review the molecular mechanisms of genetic and functional inactivation of PP2A in human cancers, with a particular focus on human acute myeloid leukemias (AML. By analyzing expression of genes encoding PP2A subunits using transcriptome sequencing, we find that PP2A dysregulation in AML is characterized by silencing and overexpression of distinct A scaffold and B regulatory subunits, respectively. We review the mechanisms of functional PP2A activation by drugs such as fingolimod, forskolin, OP449, and perphenazine. This analysis yields two non-mutually exclusive mechanisms for therapeutic PP2A re-activation: i allosteric activation of the phosphatase activity, and ii stabilization of active holo-enzyme assembly and displacement of negative regulatory factors from A and B subunits. Future studies should allow the development of specific and potent pharmacologic activators of PP2A, and definition of susceptible disease subsets based on specific mechanisms of PP2A dysregulation.

  5. Regulatory mechanisms of skeletal muscle protein turnover during exercise

    DEFF Research Database (Denmark)

    Rose, Adam John; Richter, Erik

    2009-01-01

    Skeletal muscle protein turnover is a relatively slow metabolic process that is altered by various physiological stimuli such as feeding/fasting and exercise. During exercise, catabolism of amino acids contributes very little to ATP turnover in working muscle. With regards to protein turnover......, there is now consistent data from tracer studies in rodents and humans showing that global protein synthesis is blunted in working skeletal muscle. Whether there is altered skeletal muscle protein breakdown during exercise remains unclear. The blunting of protein synthesis is believed to be mediated...... downstream of changes in intracellular Ca(2+) and energy turnover. In particular, a signaling cascade involving Ca(2+)-calmodulin-eEF2 kinase-eEF2 is implicated. The possible functional significance of altered protein turnover in working skeletal muscle during exercise is discussed. Further work...

  6. Complement regulatory protein genes in channel catfish and their involvement in disease defense response.

    Science.gov (United States)

    Jiang, Chen; Zhang, Jiaren; Yao, Jun; Liu, Shikai; Li, Yun; Song, Lin; Li, Chao; Wang, Xiaozhu; Liu, Zhanjiang

    2015-11-01

    Complement system is one of the most important defense systems of innate immunity, which plays a crucial role in disease defense responses in channel catfish. However, inappropriate and excessive complement activation could lead to potential damage to the host cells. Therefore the complement system is controlled by a set of complement regulatory proteins to allow normal defensive functions, but prevent hazardous complement activation to host tissues. In this study, we identified nine complement regulatory protein genes from the channel catfish genome. Phylogenetic and syntenic analyses were conducted to determine their orthology relationships, supporting their correct annotation and potential functional inferences. The expression profiles of the complement regulatory protein genes were determined in channel catfish healthy tissues and after infection with the two main bacterial pathogens, Edwardsiella ictaluri and Flavobacterium columnare. The vast majority of complement regulatory protein genes were significantly regulated after bacterial infections, but interestingly were generally up-regulated after E. ictaluri infection while mostly down-regulated after F. columnare infection, suggesting a pathogen-specific pattern of regulation. Collectively, these findings suggested that complement regulatory protein genes may play complex roles in the host immune responses to bacterial pathogens in channel catfish.

  7. Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element.

    Science.gov (United States)

    Mao, Grace; Brody, James P

    2007-11-09

    Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014s(-1). We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase.

  8. Bimolecular fluorescence complementation as a tool to study interactions of regulatory proteins in plant protoplasts.

    Science.gov (United States)

    Pattanaik, Sitakanta; Werkman, Joshua R; Yuan, Ling

    2011-01-01

    Protein-protein interactions are an important aspect of the gene regulation process. The expression of a gene in response to certain stimuli, within a specific cell type or at a particular developmental stage, involves a complex network of interactions between different regulatory proteins and the cis-regulatory elements present in the promoter of the gene. A number of methods have been developed to study protein-protein interactions in vitro and in vivo in plant cells, one of which is bimolecular fluorescence complementation (BiFC). BiFC is a relatively simple technique based upon the reconstitution of a fluorescent protein. The interacting protein complex can be visualized directly in a living plant cell when two non-fluorescent fragments, of an otherwise fluorescent protein, are fused to proteins found within that complex. Interaction of tagged proteins brings the two non-fluorescent fragments into close proximity and reconstitutes the fluorescent protein. In addition, the subcellular location of an interacting protein complex in the cell can be simultaneously determined. Using this approach, we have successfully demonstrated a protein-protein interaction between a R2R3 MYB and a basic helix-loop-helix MYC transcription factor related to flavonoid biosynthetic pathway in tobacco protoplasts.

  9. Effect of acute resistance exercise and sex on human patellar tendon structural and regulatory mRNA expression

    DEFF Research Database (Denmark)

    Sullivan, B.E.; Carroll, C.C.; Jemiolo, B.;

    2009-01-01

    (6 men and 6 women). Collagen type I, collagen type III, and MMP-2 were downregulated (P 0.05) 24 h after RE. All other genes remained unchanged (P > 0.05) after RE. Women had higher resting mRNA expression (P ... = 0.08) toward lower resting expression of MMP-3 than men. All other genes were not influenced (P > 0.05) by sex. Acute RE appears to stimulate a change in collagen type I, collagen type III, and MMP-2 gene regulation in the human patellar tendon. Sex influences the structural and regulatory m...... and mechanical properties, it is uncertain what structural and regulatory components contribute to these responses. We measured the mRNA expression of tendon's main fibrillar collagens (type I and type III) and the main proteoglycans (decorin, biglycan, fibromodulin, and versican) and the regulatory enzymes MMP...

  10. Physiological and regulatory effects of controlled overproduction of five cold shock proteins of Lactococcus lactis

    NARCIS (Netherlands)

    Wouters, J.A.; Mailhes, M.; Rombouts, F.M.; Vos, de W.M.; Kuipers, O.P.; Abee, T.

    2000-01-01

    The physiological and regulatory effects of overproduction of five cold shock proteins (CSPs) of Lactococcus lactis were studied. CspB, CspD, and CspE could be overproduced at high levels (up to 19␘f the total protein), whereas for CspA and CspC limited overproduction (0.3 to 0.5␘f the total protein

  11. Functional protein clusters and regulatory motifs in Hypsibius dujardini und Milnesium tardigradum

    OpenAIRE

    Förster, Frank; Liang, Chunguang; Beisser, Daniela; Frohme, Marcus; Schill, Ralph O.; Dandekar, Thomas

    2009-01-01

    Functional protein clusters and regulatory motifs do not only mediate the unique adaptation of tardigrades against extreme temperature and other harsh environmental conditions, but are important markers to distinguish species and taxonomic units. We show here in detail results of our current comparison between Hypsibius dujardini and Milnesium tardigradum. We found 50 different clusters of sequence similar proteins between both tardigrades of which 10 are tardigrade specific. Proteins of othe...

  12. A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

    Science.gov (United States)

    Yu, Chunxiao; Lopez, Carlos A; Hu, Han; Xia, Yu; Freedman, David S; Reddington, Alexander P; Daaboul, George G; Unlü, M Selim; Genco, Caroline Attardo

    2014-01-01

    The Ferric uptake regulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

  13. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    Science.gov (United States)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  14. A European pharmaceutical company initiative challenging the regulatory requirement for acute toxicity studies in pharmaceutical drug development.

    Science.gov (United States)

    Robinson, Sally; Delongeas, Jean-Luc; Donald, Elizabeth; Dreher, David; Festag, Matthias; Kervyn, Sophie; Lampo, Ann; Nahas, Kamil; Nogues, Vicente; Ockert, Deborah; Quinn, Kirsty; Old, Sally; Pickersgill, Nigel; Somers, Kev; Stark, Claudia; Stei, Peter; Waterson, Lynne; Chapman, Kathryn

    2008-04-01

    Regulatory guidelines indicate acute toxicity studies in animals are considered necessary for pharmaceuticals intended for human use. This is the only study type where lethality is mentioned as an endpoint. The studies are carried out, usually in rodents, to support marketing of new drugs and to identify the minimum lethal dose. A European initiative including 18 companies has undertaken an evidence-based review of acute toxicity studies and assessed the value of the data generated. Preclinical and clinical information was shared on 74 compounds. The analysis indicated acute toxicity data was not used to (i) terminate drugs from development (ii) support dose selection for repeat dose studies in animals or (iii) to set doses in the first clinical trials in humans. The conclusion of the working group is that acute toxicity studies are not needed prior to first clinical trials in humans. Instead, information can be obtained from other studies, which are performed at more relevant doses for humans and are already an integral part of drug development. The conclusions have been discussed and agreed with representatives of regulatory bodies from the US, Japan and Europe.

  15. Protein C and acute inflammation: a clinical and biological perspective.

    Science.gov (United States)

    Christiaans, Sarah C; Wagener, Brant M; Esmon, Charles T; Pittet, Jean Francois

    2013-10-01

    The protein C system plays an active role in modulating severe systemic inflammatory processes such as sepsis, trauma, and acute respiratory distress syndrome (ARDS) via its anticoagulant and anti-inflammatory properties. Plasma levels of activated protein C (aPC) are lower than normal in acute inflammation in humans, except early after severe trauma when high plasma levels of aPC may play a mechanistic role in the development of posttraumatic coagulopathy. Thus, following positive results of preclinical studies, a clinical trial (PROWESS) with high continuous doses of recombinant human aPC given for 4 days demonstrated a survival benefit in patients with severe sepsis. This result was not confirmed by subsequent clinical trials, including the recently published PROWESS-SHOCK trial in patients with septic shock and a phase II trial with patients with nonseptic ARDS. A possible explanation for the major difference in outcome between PROWESS and PROWESS-SHOCK trials is that lung-protective ventilation was used for the patients included in the recent PROWESS-SHOCK, but not in the original PROWESS trial. Since up to 75% of sepsis originates from the lung, aPC treatment may not have added enough to the beneficial effect of lung-protective ventilation to show lower mortality. Thus whether aPC will continue to be used to modulate the acute inflammatory response in humans remains uncertain. Because recombinant human aPC has been withdrawn from the market, a better understanding of the complex interactions between coagulation and inflammation is needed before considering the development of new drugs that modulate both coagulation and acute inflammation in humans.

  16. Elimination Half-Lives of Acute Phase Proteins in Rats and Beagle Dogs During Acute Inflammation.

    Science.gov (United States)

    Kuribayashi, Takashi; Seita, Tetsuro; Momotani, Eiichi; Yamazaki, Shunsuke; Hagimori, Kohei; Yamamoto, Shizuo

    2015-08-01

    The half-lives of typical acute phase proteins in rats and beagle dogs during acute inflammation were investigated. Acute inflammation was induced by injection of turpentine oil in rats and administration of indomethacin in beagle dogs. Serum concentrations of α2-macroglobulin (α2M) and C-reactive protein (CRP) were measured by enzyme-linked immunosorbent assay and α1-acid glycoprotein (AAG) was measured by single radial immunodiffusion. Half-life was calculated as 0.693/elimination rate constant (K). The mean half-lives in the terminal elimination phase of α2M and AAG were 68.1 and 164.8 h, respectively. The half-life of AAG was significantly longer than that of α2M. Mean half-lives in the terminal elimination phase of CRP and AAG were 161.9 and 304.4 h, respectively. The half-life of AAG was significantly longer than that of CRP in beagle dogs. No significant differences in the half-life of AAG were observed between rats and beagle dogs. Furthermore, serum concentrations in the terminal elimination phase could be simulated with the K data acquired in this study.

  17. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins Involved in a Posttranscriptional Iron Regulatory Mechanism

    Science.gov (United States)

    Figueroa-Angulo, Elisa E.; Calla-Choque, Jaeson S.; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-01-01

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

  18. Subnuclear organization and trafficking of regulatory proteins: implications for biological control and cancer.

    Science.gov (United States)

    Stein, G S; van Wijnen, A J; Stein, J L; Lian, J B; Montecino, M; Zaidi, K; Javed, A

    2000-01-01

    The regulated and regulatory components that interrelate nuclear structure and function must be experimentally established. A formidable challenge is to define further the control of transcription factor targeting to acceptor sites associated with the nuclear matrix. It will be important to determine whether acceptor proteins are associated with a pre-existing core-filament structural lattice or whether a compositely organized scaffold of regulatory factors is dynamically assembled. An inclusive model for all steps in the targeting of proteins to subnuclear sites cannot yet be proposed. However, this model must account for the apparent diversity of intranuclear targeting signals. It is also important to assess the extent to which regulatory discrimination is mediated by subnuclear domain-specific trafficking signals. Furthermore, the checkpoints that monitor subnuclear distribution of regulatory factors and the sorting steps that ensure both structural and functional fidelity of nuclear domains in which replication and expression of genes occur must be biochemically and mechanistically defined. There is emerging recognition that placement of regulatory components of gene expression must be temporally and spatially coordinated to facilitate biological control. The consequences of breaches in nuclear structure-function relationships are observed in an expanding series of diseases that include cancer [Weis et al., 1994; Rogaia et al., 1997; Yano et al., 1997; Rowley, 1998; Zeng et al., 1998; McNeil et al., 1999; Tao and Levine, 1999a] and neurological disorders [Skinner et al., 1997]. As the repertoire of architecture-associated regulatory factors and cofactors expands, workers in the field are becoming increasingly confident that nuclear organization contributes significantly to control of transcription. To gain increased appreciation for the complexities of subnuclear organization and gene regulation, we must continue to characterize mechanisms that direct

  19. Regulatory T cells reduce acute lung injury fibroproliferation by decreasing fibrocyte recruitment.

    Science.gov (United States)

    Garibaldi, Brian T; D'Alessio, Franco R; Mock, Jason R; Files, D Clark; Chau, Eric; Eto, Yoshiki; Drummond, M Bradley; Aggarwal, Neil R; Sidhaye, Venkataramana; King, Landon S

    2013-01-01

    Acute lung injury (ALI) causes significant morbidity and mortality. Fibroproliferation in ALI results in worse outcomes, but the mechanisms governing fibroproliferation remain poorly understood. Regulatory T cells (Tregs) are important in lung injury resolution. Their role in fibroproliferation is unknown. We sought to identify the role of Tregs in ALI fibroproliferation, using a murine model of lung injury. Wild-type (WT) and lymphocyte-deficient Rag-1(-/-) mice received intratracheal LPS. Fibroproliferation was characterized by histology and the measurement of lung collagen. Lung fibrocytes were measured by flow cytometry. To dissect the role of Tregs in fibroproliferation, Rag-1(-/-) mice received CD4(+)CD25(+) (Tregs) or CD4(+)CD25(-) Tcells (non-Tregs) at the time of LPS injury. To define the role of the chemokine (C-X-C motif) ligand 12 (CXCL12)-CXCR4 pathway in ALI fibroproliferation, Rag-1(-/-) mice were treated with the CXCR4 antagonist AMD3100 to block fibrocyte recruitment. WT and Rag-1(-/-) mice demonstrated significant collagen deposition on Day 3 after LPS. WT mice exhibited the clearance of collagen, but Rag-1(-/-) mice developed persistent fibrosis. This fibrosis was mediated by the sustained epithelial expression of CXCL12 (or stromal cell-derived factor 1 [SDF-1]) that led to increased fibrocyte recruitment. The adoptive transfer of Tregs resolved fibroproliferation by decreasing CXCL12 expression and subsequent fibrocyte recruitment. Blockade of the CXCL12-CXCR4 axis with AMD3100 also decreased lung fibrocytes and fibroproliferation. These results indicate a central role for Tregs in the resolution of ALI fibroproliferation by reducing fibrocyte recruitment along the CXCL12-CXCR4 axis. A dissection of the role of Tregs in ALI fibroproliferation may inform the design of new therapeutic tools for patients with ALI.

  20. DEFICIENT PROTEIN C AND PROTEIN S INDUCED ACUTE VENOUS MESENTERIC ISCHEMIA: A CASE REPORT

    Directory of Open Access Journals (Sweden)

    Darwin Britto

    2016-05-01

    Full Text Available BACKGROUND A 35 year old lady presented with unresolved severe abdominal pain and vomiting. She was diagnosed to have superior mesenteric vein thrombosis with gangrenous small bowel and multiple splenic infarcts secondary to Protein C and Protein S deficiency. She underwent emergency explorative laparotomy and extensive small bowel resection and anastomosis and splenectomy. This is to stress the importance of keeping mesenteric ischemia as an important differential diagnosis in cases of acute abdomen

  1. Violacein Treatment Modulates Acute and Chronic Inflammation through the Suppression of Cytokine Production and Induction of Regulatory T Cells.

    Science.gov (United States)

    Verinaud, Liana; Lopes, Stefanie Costa Pinto; Prado, Isabel Cristina Naranjo; Zanucoli, Fábio; Alves da Costa, Thiago; Di Gangi, Rosária; Issayama, Luidy Kazuo; Carvalho, Ana Carolina; Bonfanti, Amanda Pires; Niederauer, Guilherme Francio; Duran, Nelson; Costa, Fábio Trindade Maranhão; Oliveira, Alexandre Leite Rodrigues; Höfling, Maria Alice da Cruz; Machado, Dagmar Ruth Stach; Thomé, Rodolfo

    2015-01-01

    Inflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola), a quorum sensing metabolite from the Gram-negative bacterium Chromobacterium violaceum, has been shown to protect mice from malaria and to have beneficial effects on tumors. However, it is not known whether this drug possesses anti-inflammatory activity. In this study, we investigated whether viola administration is able to reduce acute and chronic autoimmune inflammation. For that purpose, C57BL/6 mice were intraperitoneally injected with 1 μg of LPS and were treated with viola (3.5mg/kg) via i.p. at the same time-point. Three hours later, the levels of inflammatory cytokines in the sera and phenotypical characterization of leukocytes were determined. Mice treated with viola presented a significant reduction in the production of inflammatory cytokines compared with untreated mice. Interestingly, although viola is a compound derived from bacteria, it did not induce inflammation upon administration to naïve mice. To test whether viola would protect mice from an autoimmune inflammation, Experimental Autoimmune Encephalomyelitis (EAE)-inflicted mice were given viola i.p. at disease onset, at the 10th day from immunization. Viola-treated mice developed mild EAE disease in contrast with placebo-treated mice. The frequencies of dendritic cells and macrophages were unaltered in EAE mice treated with viola. However, the sole administration of viola augmented the levels of splenic regulatory T cells (CD4+Foxp3+). We also found that adoptive transfer of viola-elicited regulatory T cells significantly reduced EAE. Our study shows, for the first time, that violacein is able to modulate acute and chronic inflammation. Amelioration relied in suppression of cytokine production (in acute inflammation) and stimulation of regulatory T cells (in chronic inflammation). New studies must be conducted in order to

  2. Violacein Treatment Modulates Acute and Chronic Inflammation through the Suppression of Cytokine Production and Induction of Regulatory T Cells.

    Directory of Open Access Journals (Sweden)

    Liana Verinaud

    Full Text Available Inflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola, a quorum sensing metabolite from the Gram-negative bacterium Chromobacterium violaceum, has been shown to protect mice from malaria and to have beneficial effects on tumors. However, it is not known whether this drug possesses anti-inflammatory activity. In this study, we investigated whether viola administration is able to reduce acute and chronic autoimmune inflammation. For that purpose, C57BL/6 mice were intraperitoneally injected with 1 μg of LPS and were treated with viola (3.5mg/kg via i.p. at the same time-point. Three hours later, the levels of inflammatory cytokines in the sera and phenotypical characterization of leukocytes were determined. Mice treated with viola presented a significant reduction in the production of inflammatory cytokines compared with untreated mice. Interestingly, although viola is a compound derived from bacteria, it did not induce inflammation upon administration to naïve mice. To test whether viola would protect mice from an autoimmune inflammation, Experimental Autoimmune Encephalomyelitis (EAE-inflicted mice were given viola i.p. at disease onset, at the 10th day from immunization. Viola-treated mice developed mild EAE disease in contrast with placebo-treated mice. The frequencies of dendritic cells and macrophages were unaltered in EAE mice treated with viola. However, the sole administration of viola augmented the levels of splenic regulatory T cells (CD4+Foxp3+. We also found that adoptive transfer of viola-elicited regulatory T cells significantly reduced EAE. Our study shows, for the first time, that violacein is able to modulate acute and chronic inflammation. Amelioration relied in suppression of cytokine production (in acute inflammation and stimulation of regulatory T cells (in chronic inflammation. New studies must be

  3. Selective translational control of the Alzheimer amyloid precursor protein transcript by iron regulatory protein-1.

    Science.gov (United States)

    Cho, Hyun-Hee; Cahill, Catherine M; Vanderburg, Charles R; Scherzer, Clemens R; Wang, Bin; Huang, Xudong; Rogers, Jack T

    2010-10-08

    Iron influx increases the translation of the Alzheimer amyloid precursor protein (APP) via an iron-responsive element (IRE) RNA stem loop in its 5'-untranslated region. Equal modulated interaction of the iron regulatory proteins (IRP1 and IRP2) with canonical IREs controls iron-dependent translation of the ferritin subunits. However, our immunoprecipitation RT-PCR and RNA binding experiments demonstrated that IRP1, but not IRP2, selectively bound the APP IRE in human neural cells. This selective IRP1 interaction pattern was evident in human brain and blood tissue from normal and Alzheimer disease patients. We computer-predicted an optimal novel RNA stem loop structure for the human, rhesus monkey, and mouse APP IREs with reference to the canonical ferritin IREs but also the IREs encoded by erythroid heme biosynthetic aminolevulinate synthase and Hif-2α mRNAs, which preferentially bind IRP1. Selective 2'-hydroxyl acylation analyzed by primer extension analysis was consistent with a 13-base single-stranded terminal loop and a conserved GC-rich stem. Biotinylated RNA probes deleted of the conserved CAGA motif in the terminal loop did not bind to IRP1 relative to wild type probes and could no longer base pair to form a predicted AGA triloop. An AGU pseudo-triloop is key for IRP1 binding to the canonical ferritin IREs. RNA probes encoding the APP IRE stem loop exhibited the same high affinity binding to rhIRP1 as occurs for the H-ferritin IRE (35 pm). Intracellular iron chelation increased binding of IRP1 to the APP IRE, decreasing intracellular APP expression in SH-SY5Y cells. Functionally, shRNA knockdown of IRP1 caused increased expression of neural APP consistent with IRP1-APP IRE-driven translation.

  4. THE REGULATORY EFFECT OF NUCLEOSIDE DIPHOSPHATE KINASE ON G-PROTEIN AND G-PROTEIN MEDIATED PHOSPHOLIPASE C

    Institute of Scientific and Technical Information of China (English)

    张德昌; 张宽仁

    1995-01-01

    The effect of nueleoside diphosphate kinase (NDPK) on the activity of guanine nueleotide regulatory protein (G-protein) mediated phospholipase C (PLC) and on the [35S ] GTPTτS binding of G-protein was investigated in this work in order to demonstrate the mechanism behind the regulation of G-protein and its effector PLC by NDPK. The stimulation of PLC in turkey erythrocyte membrane by both GTP and GTPτS indicated that the PLC stimulation was msdiated by G-protein, NDPK alone stimulated PLC activity, as well as the stimulation in the presence of GTP and GDP, in a dose-dependent manner. However, NDPK inhibited GTPτS-stimulated PLC, Furthermore, NDPK inhibited [35S] GTPτS binding of purified Gi-protein in a non-competitive manner. A hypothesis implying an important role of direct interaction of G-protein and NDPK in the regulation of their functions is suggested and discussed.

  5. Treatment with alpha-melanocyte stimulating hormone preserves calcium regulatory proteins in rat heart allografts.

    Science.gov (United States)

    Colombo, Gualtiero; Sordi, Andrea; Lonati, Caterina; Carlin, Andrea; Turcatti, Flavia; Leonardi, Patrizia; Gatti, Stefano; Catania, Anna

    2008-08-01

    Prevention of graft dysfunction is a major objective in transplantation medicine. Previous research on experimental heart transplantation indicated that treatment with the immunomodulatory peptide alpha-melanocyte stimulating hormone (alpha-MSH) improves histopathology, prolongs allograft survival, and reduces expression of the main tissue injury mediators. Because calcium-handling is critical in heart graft function, we determined the effects of transplantation injury and influences of alpha-MSH treatment on representative calcium regulatory proteins in rat heart allografts. Hearts from Brown Norway rats were transplanted heterotopically into MHC incompatible Lewis rats. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), protein kinase C epsilon (PKC epsilon), sarcoplasmic/endoplasmic reticulum calcium-ATPase 2 (SERCA2a), arrestin-beta1 (Arrb1), cholinergic receptor M2 (Chrm2), and inositol 1,4,5-triphosphate receptor 1 (InsP(3)R1) were examined in: (1) non-transplanted donor hearts; (2) allografts from saline-treated rats; and (3) allografts from rats treated with the synthetic alpha-MSH analog Nle4-DPhe7-alpha-MSH (NDP-alpha-MSH) (100 microg i.p. every 12h). Transplantation injury was associated with severe reduction in calcium regulatory protein transcription and expression level. NDP-alpha-MSH administration partly reversed inhibition of protein transcription and almost completely prevented protein loss. Finally, because certain effects of cyclic 3'-5'-adenosine monophosphate (cAMP) signaling on calcium handling in cardiac myocytes depend on activation of exchange protein directly activated by cAMP 1 (Epac1), we determined Epac1 mRNA and protein expression in heart allografts. Transplantation injury markedly reduced Epac1. NDP-alpha-MSH treatment significantly preserved both Epac1 protein and mRNA in the allografts. Administration of alpha-MSH or related melanocortins could reduce transplantation-induced dysfunction through protection of heart calcium

  6. A synthetic biology approach to self-regulatory recombinant protein production in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Dragosits Martin

    2012-03-01

    Full Text Available Abstract Background Recombinant protein production is a process of great industrial interest, with products that range from pharmaceuticals to biofuels. Since high level production of recombinant protein imposes significant stress in the host organism, several methods have been developed over the years to optimize protein production. So far, these trial-and-error techniques have proved laborious and sensitive to process parameters, while there has been no attempt to address the problem by applying Synthetic Biology principles and methods, such as integration of standardized parts in novel synthetic circuits. Results We present a novel self-regulatory protein production system that couples the control of recombinant protein production with a stress-induced, negative feedback mechanism. The synthetic circuit allows the down-regulation of recombinant protein expression through a stress-induced promoter. We used E. coli as the host organism, since it is widely used in recombinant processes. Our results show that the introduction of the self-regulatory circuit increases the soluble/insoluble ratio of recombinant protein at the expense of total protein yield. To further elucidate the dynamics of the system, we developed a computational model that is in agreement with the observed experimental data, and provides insight on the interplay between protein solubility and yield. Conclusion Our work introduces the idea of a self-regulatory circuit for recombinant protein products, and paves the way for processes with reduced external control or monitoring needs. It demonstrates that the library of standard biological parts serves as a valuable resource for initial synthetic blocks that needs to be further refined to be successfully applied in practical problems of biotechnological significance. Finally, the development of a predictive model in conjunction with experimental validation facilitates a better understanding of the underlying dynamics and can be

  7. Anti-thrombin III, Protein C, and Protein S deficiency in acute coronary syndrome

    Directory of Open Access Journals (Sweden)

    Dasnan Ismail

    2002-06-01

    Full Text Available The final most common pathway for the majority of coronary artery disease is occlusion of a coronary vessel. Under normal conditions, antithrombin III (AT III, protein C, and protein S as an active protein C cofactor, are natural anticoagulants (hemostatic control that balances procoagulant activity (thrombin antithrombin complex balance to prevent thrombosis. If the condition becomes unbalanced, natural anticoagulants and the procoagulants can lead to thrombosis. Thirty subjects with acute coronary syndrome (ACS were studied for the incidence of antithrombin III (AT III, protein C, and protein S deficiencies, and the result were compare to the control group. Among patients with ACS, the frequency of distribution of AT-III with activity < 75% were 23,3% (7 of 30, and only 6,7% ( 2 of 30 in control subject. No one of the 30 control subject have protein C activity deficient, in ACS with activity < 70% were 13,3% (4 of 30. Fifteen out of the 30 (50% control subjects had protein S activity deficiency, while protein S deficiency activity < 70% was found 73.3.% (22 out of 30. On linear regression, the deterministic coefficient of AT-III activity deficiency to the development ACS was 13,25 %, and the deterministic coefficient of protein C activity deficient to the development of ACS was 9,06 %. The cut-off point for AT-III without protein S deficiency expected to contribute to the development of vessel disease was 45%. On discriminant analysis, protein C activity deficiency posed a risk for ACS of 4,5 greater than non deficient subjects, and AT-III activity deficiency posed a risk for ACS of 3,5 times greater than non deficient subjects. On binary logistic regression, protein S activity acted only as a reinforcing factor of AT-III activity deficiency in the development of ACS. Protein C and AT III deficiency can trigger ACS, with determinant coefficients of 9,06% and 13,25% respectively. Low levels of protein C posed a greater risk of

  8. Complement and membrane-bound complement regulatory proteins as biomarkers and therapeutic targets for autoimmune inflammatory disorders, RA and SLE.

    Science.gov (United States)

    Das, Nibhriti

    2015-11-01

    Complement system is a major effecter system of the innate immunity that bridges with adaptive immunity. The system consists of about 40 humoral and cell surface proteins that include zymogens, receptors and regulators. The zymogens get activated in a cascade fashion by antigen-antibody complex, antigen alone or by polymannans, respectively, by the classical, alternative and mannose binding lectin (MBL) pathways. The ongoing research on complement regulators and complement receptors suggest key role of these proteins in the initiation, regulation and effecter mechanisms of the innate and adaptive immunity. Although, the complement system provides the first line of defence against the invading pathogens, its aberrant uncontrolled activation causes extensive self tissue injury. A large number of humoral and cell surface complement regulatory protein keep the system well-regulated in healthy individuals. Complement profiling had brought important information on the pathophysiology of several infectious and chronic inflammatory disorders. In view of the diversity of the clinical disorders involving abnormal complement activity or regulation, which include both acute and chronic diseases that affect a wide range of organs, diverse yet specifically tailored therapeutic approaches may be needed to shift complement back into balance. This brief review discusses on the complement system, its functions and its importance as biomarkers and therapeutic targets for autoimmune diseases with focus on SLE and RA.

  9. Regulatory T cell expression of CLA or α4β7 and skin or gut acute GVHD outcomes

    OpenAIRE

    Engelhardt, BG; Jagasia, M; Savani, BN; Bratcher, NL; Greer, JP; Jiang, A.; Kassim, AA; Lu, P.; Schuening, F; Yoder, SM; Rock, MT; Crowe, JE

    2010-01-01

    Regulatory T cells (Tregs) are a suppressive subset of CD4+ T lymphocytes implicated in the prevention of acute GVHD (aGVHD) after allo-SCT (ASCT). To determine whether increased frequency of Tregs with a skin-homing (cutaneous lymphocyte Ag, CLA+) or a gut-homing (α4β7+) phenotype is associated with reduced risk of skin or gut aGVHD, respectively, we quantified circulating CLA+ or α4β7+ on Tregs at the time of neutrophil engraftment in 43 patients undergoing ASCT. Increased CLA+ Tregs at eng...

  10. Dynamic control of the complement system by modulated expression of regulatory proteins.

    Science.gov (United States)

    Thurman, Joshua M; Renner, Brandon

    2011-01-01

    The complement system serves many biological functions, including the eradication of invasive pathogens and the removal of damaged cells and immune-complexes. Uncontrolled complement activation causes injury to host cells, however, so adequate regulation of the system is essential. Control of the complement system is maintained by a group of cell surface and circulating proteins referred to as complement regulatory proteins. The expression of the cell surface complement regulatory proteins varies from tissue to tissue. Furthermore, specific cell types can upregulate or downregulate the expression of these proteins in response to a variety of signals or insults. Altered regulation of the complement regulatory proteins can have important effects on local complement activation. In some circumstances this can be beneficial, such as in the setting of certain infections. In other circumstances, however, this can be a cause of complement-mediated injury of the tissue. A full understanding of the mechanisms by which the complement system is modulated at the local level can have important implications for how we diagnose and treat a wide range of inflammatory diseases.

  11. ACUTE PHASE PROTEIN AND ELECTROPHORESIS PROTEIN FRACTION VALUES FOR CAPTIVE AMERICAN FLAMINGOS (PHOENICOPTERUS RUBER).

    Science.gov (United States)

    Delk, Katie W; Wack, Raymund F; Burgdorf-Moisuk, Anne; Kass, Philip H; Cray, Carolyn

    2015-12-01

    Protein electrophoresis has recognized applications in determining the health status of various species. While reference intervals for electrophoresis have been determined for psittacine and raptor species, there are none reported for Phoenicopteriformes species. Reference intervals for haptoglobin and protein fractions obtained by electrophoresis were determined for the American flamingo (Phoenicopterus ruber) based on plasma samples from 39 captive birds. The reference intervals were as follows: haptoglobin, 0.17-0.8 mg/ml; total protein, 3.65-6.38 g/dl; prealbumin, 0.26-1.9 g/dl; albumin, 1.51-3.12 g/dl; α-1 globulin, 0.06-0.38 g/dl; α-2 globulin, 0.17-0.67 g/dl; β globulin, 0.38-1.33 g/dl; γ globulin, 0.26-0.68 g/dl; albumin : globulin ratio, 0.93-2.17. As captive flamingos often suffer from pododermatitis, feet of all flamingos were scored to determine if pododermatitis would be reflected in the acute phase proteins. Spearman rank correlation was performed on each of the protein fractions and pododermatitis scores, and only albumin had a significant correlation. This indicates that albumin, as a negative acute phase protein, may be a marker for this disease process.

  12. Iron regulatory proteins control a mucosal block to intestinal iron absorption.

    Science.gov (United States)

    Galy, Bruno; Ferring-Appel, Dunja; Becker, Christiane; Gretz, Norbert; Gröne, Hermann-Josef; Schümann, Klaus; Hentze, Matthias W

    2013-03-28

    Mammalian iron metabolism is regulated systemically by the hormone hepcidin and cellularly by iron regulatory proteins (IRPs) that orchestrate a posttranscriptional regulatory network. Through ligand-inducible genetic ablation of both IRPs in the gut epithelium of adult mice, we demonstrate that IRP deficiency impairs iron absorption and promotes mucosal iron retention via a ferritin-mediated "mucosal block." We show that IRP deficiency does not interfere with intestinal sensing of body iron loading and erythropoietic iron need, but rather alters the basal expression of the iron-absorption machinery. IRPs thus secure sufficient iron transport across absorptive enterocytes by restricting the ferritin "mucosal block" and define a basal set point for iron absorption upon which IRP-independent systemic regulatory inputs are overlaid.

  13. Iron Regulatory Proteins Control a Mucosal Block to Intestinal Iron Absorption

    Directory of Open Access Journals (Sweden)

    Bruno Galy

    2013-03-01

    Full Text Available Mammalian iron metabolism is regulated systemically by the hormone hepcidin and cellularly by iron regulatory proteins (IRPs that orchestrate a posttranscriptional regulatory network. Through ligand-inducible genetic ablation of both IRPs in the gut epithelium of adult mice, we demonstrate that IRP deficiency impairs iron absorption and promotes mucosal iron retention via a ferritin-mediated “mucosal block.” We show that IRP deficiency does not interfere with intestinal sensing of body iron loading and erythropoietic iron need, but rather alters the basal expression of the iron-absorption machinery. IRPs thus secure sufficient iron transport across absorptive enterocytes by restricting the ferritin “mucosal block” and define a basal set point for iron absorption upon which IRP-independent systemic regulatory inputs are overlaid.

  14. STUDY OF C-REACTIVE PROTEIN IN ACUTE ISCHEMIC STROKE

    Directory of Open Access Journals (Sweden)

    Medhini

    2014-06-01

    Full Text Available : Ischemic stroke is the 3rd leading cause of death after AMI and cancer. Stroke is also the leading cause of hospital admission causing disability. The study was based on 100 patients with ischemic stroke admitted to the JJM Medical College, Davangere. This study was done to estimate the role of C-reactive protein as a marker of acute inflammation following ischemic stroke and also to determine, its prognostic role, by assessing the functional outcome of patient using modified Barthel index scoring (ADL. Patients with CRP 6 mg/dl suffered severe disease with poor functional outcome. P value <0.05, significant, suggests CRP is a good tool for prognostic indicator

  15. Membrane-bound complement regulatory proteins as biomarkers and potential therapeutic targets for SLE.

    Science.gov (United States)

    Das, Nibhriti; Biswas, Bintili; Khera, Rohan

    2013-01-01

    For the last two decades, there had been remarkable advancement in understanding the role of complement regulatory proteins in autoimmune disorders and importance of complement inhibitors as therapeutics. Systemic lupus erythematosus is a prototype of systemic autoimmune disorders. The disease, though rare, is potentially fatal and afflicts women at their reproductive age. It is a complex disease with multiorgan involvement, and each patient presents with a different set of symptoms. The diagnosis is often difficult and is based on the diagnostic criteria set by the American Rheumatology Association. Presence of antinuclear antibodies and more specifically antidouble-stranded DNA indicates SLE. Since the disease is multifactorial and its phenotypes are highly heterogeneous, there is a need to identify multiple noninvasive biomarkers for SLE. Lack of validated biomarkers for SLE disease activity or response to treatment is a barrier to the efficient management of the disease, drug discovery, as well as development of new therapeutics. Recent studies with gene knockout mice have suggested that membrane-bound complement regulatory proteins (CRPs) may critically determine the sensitivity of host tissues to complement injury in autoimmune and inflammatory disorders. Case-controlled and followup studies carried out in our laboratory suggest an intimate relation between the level of DAF, MCP, CR1, and CD59 transcripts and the disease activity in SLE. Based on comparative evaluation of our data on these four membrane-bound complement regulatory proteins, we envisaged CR1 and MCP transcripts as putative noninvasive disease activity markers and the respective proteins as therapeutic targets for SLE. Following is a brief appraisal on membrane-bound complement regulatory proteins DAF, MCP, CR1, and CD59 as biomarkers and therapeutic targets for SLE.

  16. Divisome and segrosome components of Deinococcus radiodurans interact through cell division regulatory proteins.

    Science.gov (United States)

    Maurya, Ganesh K; Modi, Kruti; Misra, Hari S

    2016-08-01

    The Deinococcus radiodurans genome encodes many of the known components of divisome as well as four sets of genome partitioning proteins, ParA and ParB on its multipartite genome. Interdependent regulation of cell division and genome segregation is not understood. In vivo interactions of D. radiodurans' sdivisome, segrosome and other cell division regulatory proteins expressed on multicopy plasmids were studied in Escherichia coli using a bacterial two-hybrid system and confirmed by co-immunoprecipitation with the proteins made in E. coli. Many of these showed interactions both with the self and with other proteins. For example, DrFtsA, DrFtsZ, DrMinD, DrMinC, DrDivIVA and all four ParB proteins individually formed at least homodimers, while DrFtsA interacted with DrFtsZ, DrFtsW, DrFtsE, DrFtsK and DrMinD. DrMinD also showed interaction with DrFtsW, DrFtsE and DrMinC. Interestingly, septum site determining protein, DrDivIVA showed interactions with secondary genome ParAs as well as ParB1, ParB3 and ParB4 while DrMinC interacted with ParB1 and ParB3. PprA, a pleiotropic protein recently implicated in cell division regulation, neither interacted with divisome proteins nor ParBs but interacted at different levels with all four ParAs. These results suggest the formation of independent multiprotein complexes of 'DrFts' proteins, segrosome proteins and cell division regulatory proteins, and these complexes could interact with each other through DrMinC and DrDivIVA, and PprA in D. radiodurans.

  17. DNA-protein interaction at erythroid important regulatory elements of MEL cells by in vivo footprinting

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using ligation-mediated PCR method to study the status of DNA-protein interaction at hypersensitive site 2 of locus control Region and β maj promoter of MEL cell line before and after induction, MEL cell has been cultured and induced to differentiation by Hemin and DMSO, then the live cells have been treated with dimethyl sulfate. Ligation mediated PCR has been carried out following the chemical cleavage. The results demonstrate that before and after induction, the status of DNA-protein interaction at both hypersensitive site 2 and β maj promoter change significantly, indicating that distal regulatory elements (locus control region, hypersensitive sites) as well as proximal regulatory elements (promoter, enhancer) of β -globin gene cluster participate in the regulation of developmental specificity.

  18. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

    Science.gov (United States)

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-01-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma. PMID:27573585

  19. Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

    Energy Technology Data Exchange (ETDEWEB)

    Volkman, B.F.

    1995-02-01

    Nuclear magnetic resonance spectroscopy was used to elucidate detailed structural information for peptide and protein molecules. A small peptide was designed and synthesized, and its three-dimensional structure was calculated using distance information derived from two-dimensional NMR measurements. The peptide was used to induce antibodies in mice, and the cross-reactivity of the antibodies with a related protein was analyzed with enzyme-linked immunosorbent assays. Two proteins which are involved in regulation of transcription in bacteria were also studied. The ferric uptake regulation (Fur) protein is a metal-dependent repressor which controls iron uptake in bacteria. Two- and three-dimensional NMR techniques, coupled with uniform and selective isotope labeling allowed the nearly complete assignment of the resonances of the metal-binding domain of the Fur protein. NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a {open_quote}receiver domain{close_quote} in the family of {open_quote}two-component{close_quote} regulatory systems. Phosphorylation of the N-terminal domain of NTRC activates the initiation of transcription of aeries encoding proteins involved in nitrogen regulation. Three- and four-dimensional NMR spectroscopy methods have been used to complete the resonance assignments and determine the solution structure of the N-terminal receiver domain of the NTRC protein. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY reveals a very similar fold, with the only significant difference being the position of helix 4 relative to the rest of the protein. The determination of the structure of the NTRC receiver domain is the first step toward understanding a mechanism of signal transduction which is common to many bacterial regulatory systems.

  20. The antioxidant protein peroxiredoxin 4 is epigenetically down regulated in acute promyelocytic leukemia.

    Science.gov (United States)

    Palande, Karishma K; Beekman, Renee; van der Meeren, Lotte E; Beverloo, H Berna; Valk, Peter J M; Touw, Ivo P

    2011-01-20

    The antioxidant peroxiredoxin (PRDX) protein family comprises 6 members, which are implicated in a variety of cellular responses, including growth factor signal transduction. PRDX4 resides in the endoplasmic reticulum (ER), where it locally controls oxidative stress by reducing H(2)O(2) levels. We recently provided evidence for a regulatory function of PRDX4 in signal transduction from a myeloid growth factor receptor, the granulocyte colony-stimulating factor receptor (G-CSFR). Upon activation, the ligand-induced G-CSFR undergoes endocytosis and routes via the early endosomes where it physically interacts with ER-resident PRDX4. PRDX4 negatively regulates G-CSFR mediated signaling. Here, we investigated whether PRDX4 is affected in acute myeloid leukemia (AML); genomic alterations and expression levels of PRDX4 were investigated. We show that genomic abnormalities involving PRDX4 are rare in AML. However, we find a strong reduction in PRDX4 expression levels in acute promyelocytic leukemia (APL) compared to normal promyelocytes and different molecular subtypes of AML. Subsequently, the possible role of DNA methylation and histone modifications in silencing of PRDX4 in APLs was investigated. We show that the reduced expression is not due to methylation of the CpG island in the promoter region of PRDX4 but correlates with increased trimethylation of histone 3 lysine residue 27 (H3K27me3) and lysine residue 4 (H3K4me3) at the transcriptional start site (TSS) of PRDX4, indicative of a bivalent histone code involved in transcriptional silencing. These findings suggest that the control of G-CSF responses by the antioxidant protein PRDX4 may be perturbed in APL.

  1. The antioxidant protein peroxiredoxin 4 is epigenetically down regulated in acute promyelocytic leukemia.

    Directory of Open Access Journals (Sweden)

    Karishma K Palande

    Full Text Available The antioxidant peroxiredoxin (PRDX protein family comprises 6 members, which are implicated in a variety of cellular responses, including growth factor signal transduction. PRDX4 resides in the endoplasmic reticulum (ER, where it locally controls oxidative stress by reducing H(2O(2 levels. We recently provided evidence for a regulatory function of PRDX4 in signal transduction from a myeloid growth factor receptor, the granulocyte colony-stimulating factor receptor (G-CSFR. Upon activation, the ligand-induced G-CSFR undergoes endocytosis and routes via the early endosomes where it physically interacts with ER-resident PRDX4. PRDX4 negatively regulates G-CSFR mediated signaling. Here, we investigated whether PRDX4 is affected in acute myeloid leukemia (AML; genomic alterations and expression levels of PRDX4 were investigated. We show that genomic abnormalities involving PRDX4 are rare in AML. However, we find a strong reduction in PRDX4 expression levels in acute promyelocytic leukemia (APL compared to normal promyelocytes and different molecular subtypes of AML. Subsequently, the possible role of DNA methylation and histone modifications in silencing of PRDX4 in APLs was investigated. We show that the reduced expression is not due to methylation of the CpG island in the promoter region of PRDX4 but correlates with increased trimethylation of histone 3 lysine residue 27 (H3K27me3 and lysine residue 4 (H3K4me3 at the transcriptional start site (TSS of PRDX4, indicative of a bivalent histone code involved in transcriptional silencing. These findings suggest that the control of G-CSF responses by the antioxidant protein PRDX4 may be perturbed in APL.

  2. A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

    Directory of Open Access Journals (Sweden)

    Chunxiao Yu

    Full Text Available The Ferric uptake regulatory protein (Fur is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS, to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

  3. [The effect of extremely low doses of the novel regulatory plant proteins ].

    Science.gov (United States)

    Krasnov, M S; Margasiuk, D V; Iamskov, I A; Iamskova, V P

    2003-01-01

    Searching and study on regulatory proteins, which can keep under control the scope of important processes as like as cell adhesion, proliferation, differentiation and morphogenesis, is an actual aim of the current biochemistry. Recently we have identified S-100 proteins in plants of following species: plantain (Plantago major L.), aloe (Aloe arborescens L.), and bilberry (Vaccinum myrtillus L.). Extraction and purification of S-100 proteins gotten from these plants were performed by the method we developed earlier for adhesion proteins of animal tissues. Homogeneity of the studied plant proteins was evaluated and confirmed by HPLC and SDS-electrophoresis in PAAG. Both, plant and animal proteins have appeared to be biologically active at extremely low doses. The tests were performed by adhesiometrical method in short-term tissue culture of mouse's liver in vitro. As a result it was established that the plant proteins insert a membranotropic effect being added in extremely low doses, corresponding to 10(-10)-10(-13) mg/ml. Keeping in mind that the plantain is well known remedy for wound protection and healing, in several experiments we studied the biological effect of plant S-100 proteins on animal cells. It was found that S-100 proteins obtained from plantain influences proliferation of human fibroblasts in vitro. It was found that after the treatment with this protein in low doses the cell growth rate increases essentially.

  4. Report of the European Society of Cardiology Cardiovascular Round Table regulatory workshop update of the evaluation of new agents for the treatment of acute coronary syndrome

    DEFF Research Database (Denmark)

    Bueno, Héctor; de Graeff, Pieter; Richard-Lordereau, Isabelle

    2016-01-01

    Regulatory authorities interpret the results of randomized controlled trials according to published principles. The European Medicines Agency (EMA) is planning a revision of the 2000 and 2003 guidance documents on clinical investigation of new medicinal products for the treatment of acute coronary...... researchers, trialists, European and US regulators, and pharmaceutical industry researchers. Specific questions in four key areas were selected as priorities for changes in regulatory guidance: patient selection, endpoints, methodologic issues and issues related to the research for novel agents. Patients...

  5. Proteomic shifts in embryonic stem cells with gene dose modifications suggest the presence of balancer proteins in protein regulatory networks.

    Directory of Open Access Journals (Sweden)

    Lei Mao

    Full Text Available Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of "balancer" proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the "elasticity" of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions.

  6. Lanosterol metabolism and sterol regulatory element binding protein (SREBP) expression in male germ cell maturation.

    Science.gov (United States)

    Fon Tacer, Klementina; Kalanj-Bognar, Svjetlana; Waterman, Michael R; Rozman, Damjana

    2003-06-01

    Expression of genes involved in cholesterol biosynthesis in male germ cells is insensitive to the negative cholesterol feedback regulation, in contrast to cholesterol level-sensitive/sterol regulatory element binding protein (SREBP)-dependent gene regulation in somatic cells. The role of sterol regulatory element binding proteins in spermatogenic cells was an enigma until recently, when a soluble, 55kDa cholesterol-insensitive form of SREBP2 (SREBP2gc) was discovered [Mol. Cell. Endocrinol. 22 (2002) 8478], being translated from a germ cell-specific SREBP2 mRNA. Our RT-PCR results also show that SREBP2 as well as SREBP1c mRNAs are detectable in prepubertal and postpubertal male germ cells while SREBP1a is not detected. Surprisingly, three SREBP2 immunoreactive proteins (72, 63 and 55kDa), that are not present in mouse liver nuclei, reside in testis nuclei of prepubertal and adult mice. The 55kDa protein is likely SREBP2gc, the other two isoforms are novel. HPLC measurements in liver and testes of fasted prepubertal and postpubertal mice showed no significant difference in cholesterol level. However, FF-MAS and lanosterol/testis-meiosis activating sterol (T-MAS) intermediates that are detectable mainly in testes, increase in fasted postpubertal mice which coincides well with the elevated level of 68kDa SREBP2. Similar to SREBP2gc, the two novel SREBP2 immunoreactive proteins seem to be insensitive to the level of cholesterol.

  7. Peripheral nerve proteins as potential autoantigens in acute and chronic inflammatory demyelinating polyneuropathies.

    Science.gov (United States)

    Lim, Jia Pei; Devaux, Jérôme; Yuki, Nobuhiro

    2014-10-01

    Guillain-Barré syndrome is classified into acute inflammatory demyelinating polyneuropathy and acute motor axonal neuropathy. Whereas autoantibodies to GM1 or GD1a induce the development of acute motor axonal neuropathy, pathogenic autoantibodies have yet to be identified in acute inflammatory demyelinating polyneuropathy and chronic inflammatory demyelinating polyneuropathy. This review highlights the importance of autoantibodies to peripheral nerve proteins in the physiopathology of acute and chronic inflammatory demyelinating polyneuropathies. Moreover, we listed up other potential antigens, which may become helpful biomarkers for acquired, dysimmune demyelinating neuropathies based on their critical functions during myelination and their implications in hereditary demyelinating neuropathies.

  8. Cytoplasmic dynein and its regulatory proteins in Golgi pathology in nervous system disorders

    Directory of Open Access Journals (Sweden)

    Dick eJaarsma

    2015-10-01

    Full Text Available The Golgi apparatus is a dynamic organelle involved in processing and sorting of lipids and proteins. In neurons, the Golgi apparatus is important for the development of axons and dendrites and maintenance of their highly polarized morphology. The motor protein complex cytoplasmic dynein has an important role in Golgi apparatus positioning and function. Together with dynactin and other regulatory factors it drives microtubule minus-end directed motility of Golgi membranes. Inhibition of dynein results in fragmentation and dispersion of the Golgi ribbon in the neuronal cell body, resembling the Golgi abnormalities observed in some neurodegenerative disorders, in particular motor neuron diseases. Mutations in dynein and its regulatory factors, including the dynactin subunit p150Glued, BICD2 and Lis-1, are associated with several human nervous system disorders, including cortical malformation and motor neuropathy. Here we review the role of dynein and its regulatory factors in Golgi function and positioning, and the potential role of dynein malfunction in causing Golgi apparatus abnormalities in nervous system disorders.

  9. RNA regulatory networks diversified through curvature of the PUF protein scaffold.

    Science.gov (United States)

    Wilinski, Daniel; Qiu, Chen; Lapointe, Christopher P; Nevil, Markus; Campbell, Zachary T; Tanaka Hall, Traci M; Wickens, Marvin

    2015-09-14

    Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p-RNA complexes reveal that the protein scaffold presents an exceptionally flat and extended interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks.

  10. Acute phase proteins response to feed deprivation in broiler chickens.

    Science.gov (United States)

    Najafi, P; Zulkifli, I; Soleimani, A F; Goh, Y M

    2016-04-01

    Feed deprivation in poultry farming imposes some degree of stress to the birds, and adversely affects their well -being. Serum levels of acute phase proteins (APP) are potential physiological indicators of stress attributed to feed deprivation. However, it has not been determined how long it takes for a measurable APP response to stressors to occur in avian species. An experiment was designed to delineate the APP and circulating levels of corticosterone responses in commercial broiler chickens to feed deprivation for 30 h. It was hypothesized that feed deprivation would elicit both APP and corticosterone (CORT) reactions within 30 h that is probably associated with stress of hunger. Twenty-one day old birds were subjected to one of 5 feed deprivation periods: 0 (ad libitum, AL), 6, 12, 18, 24, and 30 h. Upon completion of the deprivation period, blood samples were collected to determine serum CORT, ovotransferrin (OVT), α1-acid glycoprotein (AGP), and ceruloplasmin (CP) concentrations. Results showed that feed deprivation for 24 h or more caused a marked elevation in CORT (P=0.002 and Pstressful condition than CORT response in assessing the well-being of broiler chickens.

  11. Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.

    OpenAIRE

    Breunig, K D; Kuger, P

    1987-01-01

    As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wil...

  12. Pancreatic islets engineered with SA-FasL protein establish robust localized tolerance by inducing regulatory T cells in mice.

    Science.gov (United States)

    Yolcu, Esma S; Zhao, Hong; Bandura-Morgan, Laura; Lacelle, Chantale; Woodward, Kyle B; Askenasy, Nadir; Shirwan, Haval

    2011-12-01

    Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL-engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL-engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and maintained by CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of type 1 diabetes.

  13. Pancreatic Islets Engineered with SA-FasL Protein Establish Robust Localized Tolerance by Inducing T Regulatory Cells in Mice

    Science.gov (United States)

    Yolcu, Esma S; Zhao, Hong; Bandura-Morgan, Laura; Lacelle, Chantale; Woodward, Kyle B; Askenasy, Nadir; Shirwan, Haval

    2011-01-01

    Allogeneic islet transplantation is an important therapeutic approach for the treatment of T1D. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic T effector cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating T effector cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of FasL protein chimeric with streptavidin (SA-FasL), and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for over a week in vitro. SA-FasL-engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL-engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% C57BL/6 recipients. Tolerance was initiated and maintained by CD4+CD25+FoxP3+ T regulatory (Treg) cells as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of T1D. PMID:22068235

  14. Structure of dual function iron regulatory protein 1 complexed with ferritin IRE-RNA

    Energy Technology Data Exchange (ETDEWEB)

    Walden, William E.; Selezneva, Anna I.; Dupuy, Jérôme; Volbeda, Anne; Fontecilla-Camps, Juan C.; Theil, Elizabeth C.; Volz1, Karl (IBS); (CHORI); (UIC)

    2011-07-27

    Iron regulatory protein 1 (IRP1) binds iron-responsive elements (IREs) in messenger RNAs (mRNAs), to repress translation or degradation, or binds an iron-sulfur cluster, to become a cytosolic aconitase enzyme. The 2.8 angstrom resolution crystal structure of the IRP1:ferritin H IRE complex shows an open protein conformation compared with that of cytosolic aconitase. The extended, L-shaped IRP1 molecule embraces the IRE stem-loop through interactions at two sites separated by {approx}30 angstroms, each involving about a dozen protein:RNA bonds. Extensive conformational changes related to binding the IRE or an iron-sulfur cluster explain the alternate functions of IRP1 as an mRNA regulator or enzyme.

  15. High mobility group box 1 protein as a late-acting mediator of acute lung inflammation.

    Science.gov (United States)

    Lutz, Waldemar; Stetkiewicz, Jan

    2004-01-01

    Acute inflammatory lung injury is often a delayed complication of critical illness and is associated with increased mortality. High mobility group box 1 (HMGB1) protein, in addition to its role as a transcriptional regulator factor, has been identified as a late mediator of endotoxin lethality and might be also involved in the development and progression of acute lung injury. HMGB1 protein itself can cause an acute inflammatory response manifested by increased production of proinflammatory cytokines and neutrophil accumulation. The delayed kinetics of HMGB1 protein release indicate that this protein is a distal mediator of acute inflamatory lung injury. Anti-HMGB1 protein antibodies attenuated endotoxin-induced lung injury, but not the early release of TNF-alpha and IL-1beta, indicating that HMGB1 protein is a late mediator of endotoxin-induced acute lung injury. HMGB1 protein is not released by apoptotic cells but is passively released by necrotic or damaged somatic and immune cells and it functions as a major stimulus of necrosis-induced inflammation. HMGB1 protein is also released by activated monocytes/macrophages and induces delayed and biphasic release of proinflammatory mediators from these cells. HMGB1 protein failed to stimulate cytokines release in lymphocytes, indicating that cellular stimulation is specific. We would like to suggest that HMGB1 protein may be also a primary mediator of the inflammatory responses to lung cells injury caused by toxic environmental chemicals.

  16. Role of complement and complement regulatory proteins in the complications of diabetes.

    Science.gov (United States)

    Ghosh, Pamela; Sahoo, Rupam; Vaidya, Anand; Chorev, Michael; Halperin, Jose A

    2015-06-01

    It is well established that the organ damage that complicates human diabetes is caused by prolonged hyperglycemia, but the cellular and molecular mechanisms by which high levels of glucose cause tissue damage in humans are still not fully understood. The prevalent hypothesis explaining the mechanisms that may underlie the pathogenesis of diabetes complications includes overproduction of reactive oxygen species, increased flux through the polyol pathway, overactivity of the hexosamine pathway causing intracellular formation of advanced glycation end products, and activation of protein kinase C isoforms. In addition, experimental and clinical evidence reported in past decades supports a strong link between the complement system, complement regulatory proteins, and the pathogenesis of diabetes complications. In this article, we summarize the body of evidence that supports a role for the complement system and complement regulatory proteins in the pathogenesis of diabetic vascular complications, with specific emphasis on the role of the membrane attack complex (MAC) and of CD59, an extracellular cell membrane-anchored inhibitor of MAC formation that is inactivated by nonenzymatic glycation. We discuss a pathogenic model of human diabetic complications in which a combination of CD59 inactivation by glycation and hyperglycemia-induced complement activation increases MAC deposition, activates pathways of intracellular signaling, and induces the release of proinflammatory, prothrombotic cytokines and growth factors. Combined, complement-dependent and complement-independent mechanisms induced by high glucose promote inflammation, proliferation, and thrombosis as characteristically seen in the target organs of diabetes complications.

  17. Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population.

    Science.gov (United States)

    Depauw, Sarah; Delanghe, Joris; Whitehouse-Tedd, Katherine; Kjelgaard-Hansen, Mads; Christensen, Michelle; Hesta, Myriam; Tugirimana, Pierrot; Budd, Jane; Dermauw, Veronique; Janssens, Geert P J

    2014-09-01

    Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type. Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations within reference ranges for healthy domestic cats. In contrast, unhealthy cheetahs had higher (P cheetahs suffering from chronic kidney disease were significantly greater compared to the reportedly healthy cheetahs. Our study indicates that serum proteins in the cheetah can be analyzed by routine capillary electrophoresis, whereas acute-phase proteins can be measured using available immunoassays or non-species-specific techniques, which are also likely to be applicable in other exotic felids. Moreover, results suggest that serum amyloid A and haptoglobin are important acute-phase proteins in the diseased cheetah and highlight the need to evaluate their role as early-onset markers for disease.

  18. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions

    DEFF Research Database (Denmark)

    Luo, Yonglun; Friis, Jenny Blechingberg; Fernandes, Ana Miguel;

    2015-01-01

    IP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression...... at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. Conclusions The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes...

  19. Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

    OpenAIRE

    Andreas Bitter; Nüssler, Andreas K.; Thasler, Wolfgang E.; Kathrin Klein; Zanger, Ulrich M.; Matthias Schwab; Oliver Burk

    2015-01-01

    Background/Aims: Sterol regulatory element-binding protein (SREBP) 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human li...

  20. Spatial proximity statistics suggest a regulatory role of protein phosphorylation on compound binding.

    Science.gov (United States)

    Korkuć, Paula; Walther, Dirk

    2016-05-01

    Phosphorylation is an important post-translational modification that regulates protein function by the attachment of negatively charged phosphate groups to phosphorylatable amino acid residues. As a mode of action, an influence of phosphorylation on the binding of compounds to proteins has been discussed and described for a number of proteins in the literature. However, a systematic statistical survey probing for enriched phosphorylation sites close to compound binding sites in support of this notion and with properly chosen random reference distributions has not been presented yet. Using high-resolution protein structures from the Protein Data Bank including their co-crystallized non-covalently bound compounds and experimentally determined phosphorylation sites, we analyzed the pairwise distance distributions of phosphorylation and compound binding sites on protein surfaces. We found that phosphorylation sites are indeed located at significantly closer distances to compounds than expected by chance holding true specifically also for the subset of compound binding sites serving as catalytic sites of metabolic reactions. This tendency was particularly evident when treating phosphorylation sites as collective sets supporting the relevance of phosphorylation hotspots. Interestingly, phosphorylation sites were found to be closer to negatively charged than to positively charged compounds suggesting a stronger modulation of the binding of negatively charged compounds in dependence on phosphorylation status than on positively charged compounds. The enrichment of phosphorylation sites near compound binding sites confirms a regulatory role of phosphorylation in compound binding and provides a solid statistical basis for the literature-reported selected events.

  1. Role of Glucokinase in the Subcellular Localization of Glucokinase Regulatory Protein

    Directory of Open Access Journals (Sweden)

    Ling Jin

    2015-04-01

    Full Text Available Glucokinase (GCK is the rate-limiting enzyme of liver glucose metabolism. Through protein-protein interactions, glucokinase regulatory protein (GCKR post-transcriptionally regulates GCK function in the liver, and causes its nuclear localization. However the role of GCK in regulating GCKR localization is unknown. In the present study, using in vitro and in vivo models, we examined the levels of GCK and GCKR, and their subcellular localization. We found that total cellular levels of GCKR did not vary in the in vivo models, but its subcellular localization did. In animals with normal levels of GCK, GCKR is mainly localized to the nuclei of hepatocytes. In seven-day old rats and liver-specific Gck gene knockout mice (animals that lack or have reduced levels of GCK protein, GCKR was found primarily in the cytoplasm. The interaction of GCK with GCKR was further examined using in vitro models where we varied the levels of GCK and GCKR. Varying the level of GCK protein had no effect on total cellular GCKR protein levels. Taken together, our results indicate that GCK is important for the localization of GCKR to the nucleus and raises the possibility that GCKR may have functions in addition to those regulating GCK activity in the cytoplasm.

  2. Reference intervals for acute phase protein and serum protein electrophoresis values in captive Asian elephants (Elephas maximus).

    Science.gov (United States)

    Isaza, Ramiro; Wiedner, Ellen; Hiser, Sarah; Cray, Carolyn

    2014-09-01

    Acute phase protein (APP) immunoassays and serum protein electrophoresis (SPEP) are assays for evaluating the inflammatory response and have use as diagnostic tools in a variety of species. Acute phase proteins are markers of inflammation that are highly conserved across different species while SPEP separates and quantifies serum protein fractions based on their physical properties. In the current study, serum samples from 35 clinically healthy Asian elephants (Elephas maximus) were analyzed using automated assays for C-reactive protein, serum amyloid A, and haptoglobin and SPEP. Robust methods were used to generate reference intervals for the APPs: C-reactive protein (1.3-12.8 mg/l), serum amyloid A (0-47.5 mg/l), and haptoglobin (0-1.10 mg/ml). In addition, SPEP was performed on these samples to establish reference intervals for each protein fraction. A combination of APPs and SPEP measurements are valuable adjunctive diagnostic tools in elephant health care.

  3. Proliferation of transformed somatotroph cells related to low or absent expression of protein kinase a regulatory subunit 1A protein.

    Science.gov (United States)

    Lania, Andrea G; Mantovani, Giovanna; Ferrero, Stefano; Pellegrini, Caterina; Bondioni, Sara; Peverelli, Erika; Braidotti, Paola; Locatelli, Marco; Zavanone, Mario L; Ferrante, Emanuela; Bosari, Silvano; Beck-Peccoz, Paolo; Spada, Anna

    2004-12-15

    The two regulatory subunits (R1 and R2) of protein kinase A (PKA) are differentially expressed in cancer cell lines and exert diverse roles in growth control. Recently, mutations of the PKA regulatory subunit 1A gene (PRKAR1A) have been identified in patients with Carney complex. The aim of this study was to evaluate the expression of the PKA regulatory subunits R1A, R2A, and R2B in a series of 30 pituitary adenomas and the effects of subunit activation on cell proliferation. In these tumors, neither mutation of PRKAR1A nor loss of heterozygosity was identified. By real-time PCR, mRNA of the three subunits was detected in all of the tumors, R1A being the most represented in the majority of samples. By contrast, immunohistochemistry documented low or absent R1A levels in all tumors, whereas R2A and R2B were highly expressed, thus resulting in an unbalanced R1/R2 ratio. The low levels of R1A were, at least in part, due to proteasome-mediated degradation. The effect of the R1/R2 ratio on proliferation was assessed in GH3 cells, which showed a similar unbalanced pattern of R subunits expression, and in growth hormone-secreting adenomas. The R2-selective cAMP analog 8-Cl cAMP and R1A RNA silencing, stimulated cell proliferation and increased Cyclin D1 expression, respectively, in human and rat adenomatous somatotrophs. These data show that a low R1/R2 ratio promoted proliferation of transformed somatotrophs and are consistent with the Carney complex model in which R1A inactivating mutations further unbalance this ratio in favor of R2 subunits. These results suggest that low expression of R1A protein may favor cAMP-dependent proliferation of transformed somatotrophs.

  4. The Acute-Phase Proteins Serum Amyloid A and C Reactive Protein in Transudates and Exudates

    Science.gov (United States)

    Okino, Alessandra M.; Bürger, Cristiani; Cardoso, Jefferson R.; Lavado, Edson L.; Lotufo, Paulo A.; Campa, Ana

    2006-01-01

    The distinction between exudates and transudates is very important in the patient management. Here we evaluate whether the acute-phase protein serum amyloid A (SAA), in comparison with C reactive protein (CRP) and total protein (TP), can be useful in this discrimination. CRP, SAA, and TP were determined in 36 exudate samples (27 pleural and 9 ascitic) and in 12 transudates (9 pleural and 3 ascitic). CRP, SAA, and TP were measured. SAA present in the exudate corresponded to 10% of the amount found in serum, that is, the exudate/serum ratio (E/S) was 0.10 ± 0.13. For comparison, the exudate/serum ratio for CRP and TP was 0.39 ± 0.37 and 0.68 ± 0.15, respectively. There was a strong positive correlation between serum and exudate SAA concentration (r = 0.764;p < 0.0001). The concentration of SAA in transudates was low and did not overlap with that found in exudates (0.02-0.21 versus 0.8–360.5 g/mL). SAA in pleural and ascitic exudates results mainly from leakage of the serum protein via the inflamed membrane. A comparison of the E/S ratio of SAA and CRP points SAA as a very good marker in discriminating between exudates and transudates. PMID:16864904

  5. Identification of Functional Regulatory Residues of the β-Lactam Inducible Penicillin Binding Protein in Methicillin-Resistant Staphylococcus aureus

    OpenAIRE

    Mbah, Andreas N.; Isokpehi, Raphael D

    2013-01-01

    Resistance to methicillin by Staphylococcus aureus is a persistent clinical problem worldwide. A mechanism for resistance has been proposed in which methicillin resistant Staphylococcus aureus (MRSA) isolates acquired a new protein called β -lactam inducible penicillin binding protein (PBP-2′). The PBP-2′ functions by substituting other penicillin binding proteins which have been inhibited by β -lactam antibiotics. Presently, there is no structural and regulatory information on PBP-2′ protein...

  6. Activated protein C in the treatment of acute lung injury and acute respiratory distress syndrome

    NARCIS (Netherlands)

    A.D. Cornet; G.P. van Nieuw Amerongen; A. Beishuizen; M.J. Schultz; A.R.J. Girbes; A.B.J. Groeneveld

    2009-01-01

    Background: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) frequently necessitate mechanical ventilation in the intensive care unit. The syndromes have a high mortality rate and there is at present no treatment specifically directed at the underlying pathogenesis. Central in

  7. Effector and regulatory T cells in patients with acute optic neuritis

    DEFF Research Database (Denmark)

    Tsakiri, Anna; Kjærsgaard, Erik; Grigoriadis, Nikolaos;

    2012-01-01

    Optic neuritis (ON) is an autoimmune acute demyelinating disease of the optic nerve and may occur in patients with confirmed multiple sclerosis (MS) or as a clinically isolated syndrome. T lymphocytes play a central role in the pathogenesis of MS. The phenotype of different T cell subsets is usua...... is usually characterized by the expression of distinct cell surface receptors such as CD45RA, CD45RO, CCR7, CD27 and CD28. The aim of this study was to characterize the phenotype of distinct subsets of CD4 and CD8 T cells in patients with isolated ON....

  8. Protein phosphatase 2A regulatory subunit B56α limits phosphatase activity in the heart.

    Science.gov (United States)

    Little, Sean C; Curran, Jerry; Makara, Michael A; Kline, Crystal F; Ho, Hsiang-Ting; Xu, Zhaobin; Wu, Xiangqiong; Polina, Iuliia; Musa, Hassan; Meadows, Allison M; Carnes, Cynthia A; Biesiadecki, Brandon J; Davis, Jonathan P; Weisleder, Noah; Györke, Sandor; Wehrens, Xander H; Hund, Thomas J; Mohler, Peter J

    2015-07-21

    Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme composed of a catalytic, scaffolding, and regulatory subunit. In the heart, PP2A activity is requisite for cardiac excitation-contraction coupling and central in adrenergic signaling. We found that mice deficient in the PP2A regulatory subunit B56α (1 of 13 regulatory subunits) had altered PP2A signaling in the heart that was associated with changes in cardiac physiology, suggesting that the B56α regulatory subunit had an autoinhibitory role that suppressed excess PP2A activity. The increase in PP2A activity in the mice with reduced B56α expression resulted in slower heart rates and increased heart rate variability, conduction defects, and increased sensitivity of heart rate to parasympathetic agonists. Increased PP2A activity in B56α(+/-) myocytes resulted in reduced Ca(2+) waves and sparks, which was associated with decreased phosphorylation (and thus decreased activation) of the ryanodine receptor RyR2, an ion channel on intracellular membranes that is involved in Ca(2+) regulation in cardiomyocytes. In line with an autoinhibitory role for B56α, in vivo expression of B56α in the absence of altered abundance of other PP2A subunits decreased basal phosphatase activity. Consequently, in vivo expression of B56α suppressed parasympathetic regulation of heart rate and increased RyR2 phosphorylation in cardiomyocytes. These data show that an integral component of the PP2A holoenzyme has an important inhibitory role in controlling PP2A enzyme activity in the heart.

  9. iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness

    Science.gov (United States)

    Wu, Guiqin; Shi, Fengying; Liu, Aiqiao; Yang, Ning

    2016-01-01

    Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57) would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 (SLC25A5) and down-regulated translocator protein (TSPO) would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF). In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation. PMID:28006025

  10. Malignant mixed Mullerian tumors of the uterus: histopathological evaluation of cell cycle and apoptotic regulatory proteins

    Directory of Open Access Journals (Sweden)

    Senger Jenna-Lynn B

    2010-07-01

    Full Text Available Abstract Aim The aim of our study was to evaluate survival outcomes in malignant mixed Mullerian tumors (MMMT of the uterus with respect to the role of cell cycle and apoptotic regulatory proteins in the carcinomatous and sarcomatous components. Methods 23 cases of uterine MMMT identified from the Saskatchewan Cancer Agency (1970-1999 were evaluated. Immunohistochemical expression of Bad, Mcl-1, bcl-x, bak, mdm2, bax, p16, p21, p53, p27, EMA, Bcl-2, Ki67 and PCNA was correlated with clinico-pathological data including survival outcomes. Results Histopathological examination confirmed malignant epithelial component with homologous (12 cases and heterologous (11 cases sarcomatous elements. P53 was strongly expressed (70-95% in 15 cases and negative in 5 cases. The average survival in the p53+ve cases was 3.56 years as opposed to 8.94 years in p53-ve cases. Overexpression of p16 and Mcl-1 were observed in patients with longer survival outcomes (> 2 years. P16 and p21 were overexpressed in the carcinomatous and sarcomatous elements respectively. Cyclin-D1 was focally expressed only in the carcinomatous elements. Conclusions Our study supports that a cell cycle and apoptotic regulatory protein dysregulation is an important pathway for tumorigenesis and b p53 is an important immunoprognostic marker in MMMT of the uterus.

  11. Physiological and Regulatory Effects of Controlled Overproduction of Five Cold Shock Proteins of Lactococcus lactis MG1363

    NARCIS (Netherlands)

    Wouters, Jeroen A.; Mailhes, Marielle; Rombouts, Frank M.; Vos, Willem M. de; Kuipers, Oscar P.; Abee, Tjakko

    2000-01-01

    The physiological and regulatory effects of overproduction of five cold shock proteins (CSPs) of Lactococcus lactis were studied. CspB, CspD, and CspE could be overproduced at high levels (up to 19% of the total protein), whereas for CspA and CspC limited overproduction (0.3 to 0.5% of the total pro

  12. Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin

    Directory of Open Access Journals (Sweden)

    Rotwein Peter

    2008-04-01

    Full Text Available Abstract Background Repulsive guidance molecule c (RGMc or hemojuvelin, a glycosylphosphatidylinositol-linked glycoprotein expressed in liver and striated muscle, plays a central role in systemic iron balance. Inactivating mutations in the RGMc gene cause juvenile hemochromatosis (JH, a rapidly progressing iron storage disorder with severe systemic manifestations. RGMc undergoes complex biosynthetic steps leading to membrane-bound and soluble forms of the protein, including both 50 and 40 kDa single-chain species. Results We now show that pro-protein convertases (PC are responsible for conversion of 50 kDa RGMc to a 40 kDa protein with a truncated COOH-terminus. Unlike related molecules RGMa and RGMb, RGMc encodes a conserved PC recognition and cleavage site, and JH-associated RGMc frame-shift mutants undergo COOH-terminal cleavage only if this site is present. A cell-impermeable peptide PC inhibitor blocks the appearance of 40 kDa RGMc in extra-cellular fluid, as does an engineered mutation in the conserved PC recognition sequence, while the PC furin cleaves 50 kDa RGMc in vitro into a 40 kDa molecule with an intact NH2-terminus. Iron loading reduces release of RGMc from the cell membrane, and diminishes accumulation of the 40 kDa species in cell culture medium. Conclusion Our results define a role for PCs in the maturation of RGMc that may have implications for the physiological actions of this critical iron-regulatory protein.

  13. Calcium/calmodulin-dependent protein kinase IV mediates acute nicotine-induced antinociception in acute thermal pain tests.

    Science.gov (United States)

    Jackson, Kia J; Damaj, Mohamad I

    2013-12-01

    Calcium-activated second messengers such as calcium/calmodulin-dependent protein kinase II have been implicated in drug-induced antinociception. The less abundant calcium-activated second messenger, calcium/calmodulin-dependent protein kinase IV (CaMKIV), mediates emotional responses to pain and tolerance to morphine analgesia but its role in nicotine-mediated antinociception is currently unknown. The goal of this study was to evaluate the role of CaMKIV in the acute effects of nicotine, primarily acute nicotine-induced antinociception. CaMKIV knockout (-/-), heterozygote (+/-), and wild-type (+/+) mice were injected with various doses of nicotine and evaluated in a battery of tests, including the tail-flick and hot-plate tests for antinociception, body temperature, and locomotor activity. Our results show a genotype-dependent reduction in tail-flick and hot-plate latency in CaMKIV (+/-) and (-/-) mice after acute nicotine treatment, whereas no difference was observed between genotypes in the body temperature and locomotor activity assessments. The results of this study support a role for CaMKIV in acute nicotine-induced spinal and supraspinal pain mechanisms, and further implicate involvement of calcium-dependent mechanisms in drug-induced antinociception.

  14. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues

    Science.gov (United States)

    Suzuki, Takashi; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5′-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5′-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity. PMID:27256115

  15. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yeming; Opperman, Laura; Wickens, Marvin; Tanaka Hall, Traci M. (NIH); (UW)

    2011-11-02

    Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

  16. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yeming; Opperman, Laura; Wickens, Marvin; Tanaka Hall, Traci M.; (NIH); (UW)

    2010-08-19

    Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

  17. Mutations in complement regulatory proteins predispose to preeclampsia: a genetic analysis of the PROMISSE cohort.

    Directory of Open Access Journals (Sweden)

    Jane E Salmon

    2011-03-01

    Full Text Available BACKGROUND: Pregnancy in women with systemic lupus erythematosus (SLE or antiphospholipid antibodies (APL Ab--autoimmune conditions characterized by complement-mediated injury--is associated with increased risk of preeclampsia and miscarriage. Our previous studies in mice indicate that complement activation targeted to the placenta drives angiogenic imbalance and placental insufficiency. METHODS AND FINDINGS: We use PROMISSE, a prospective study of 250 pregnant patients with SLE and/or APL Ab, to test the hypothesis in humans that impaired capacity to limit complement activation predisposes to preeclampsia. We sequenced genes encoding three complement regulatory proteins--membrane cofactor protein (MCP, complement factor I (CFI, and complement factor H (CFH--in 40 patients who had preeclampsia and found heterozygous mutations in seven (18%. Five of these patients had risk variants in MCP or CFI that were previously identified in atypical hemolytic uremic syndrome, a disease characterized by endothelial damage. One had a novel mutation in MCP that impairs regulation of C4b. These findings constitute, to our knowledge, the first genetic defects associated with preeclampsia in SLE and/or APL Ab. We confirmed the association of hypomorphic variants of MCP and CFI in a cohort of non-autoimmune preeclampsia patients in which five of 59 were heterozygous for mutations. CONCLUSION: The presence of risk variants in complement regulatory proteins in patients with SLE and/or APL Ab who develop preeclampsia, as well as in preeclampsia patients lacking autoimmune disease, links complement activation to disease pathogenesis and suggests new targets for treatment of this important public health problem. STUDY REGISTRATION: ClinicalTrials.gov NCT00198068.

  18. Acute differential effects of dietary protein quality on postprandial lipemia in obese non-diabetic subjects

    DEFF Research Database (Denmark)

    Holmer-Jensen, Jens; Mortensen, Lene S; Astrup, Arne

    2013-01-01

    Non-fasting triglyceridemia is much closer associated to cardiovascular risk compared to fasting triglyceridemia. We hypothesized that there would be acute differential effects of four common dietary proteins (cod protein, whey isolate, gluten, and casein) on postprandial lipemia in obese non......-diabetic subjects. To test the hypothesis we conducted a randomized, acute clinical intervention study with crossover design. We supplemented a fat rich mixed meal with one of four dietary proteins i.e. cod protein, whey protein, gluten or casein. Eleven obese non-diabetic subjects (age: 40-68, body mass index: 30.......3-42.0 kg/m(2)) participated and blood samples were drawn in the 8-h postprandial period. Supplementation of a fat rich mixed meal with whey protein caused lower postprandial lipemia (P = .048) compared to supplementation with cod protein and gluten. This was primarily due to lower triglyceride...

  19. Volume regulatory loss of Na, Cl, and K from rat brain during acute hyponatremia

    Energy Technology Data Exchange (ETDEWEB)

    Melton, J.E.; Patlak, C.S.; Pettigrew, K.D.; Cserr, H.F.

    1987-04-01

    This study quantitatively evaluates the contribution of tissue Na, Cl, and K loss to brain volume regulation during acute dilutional hyponatremia (DH) and examines the mechanism of Na loss. DH was produced in pentobarbital sodium-anesthetized rats by intraperitoneal infusion of distilled water and brain water and electrolytes analyzed 30 min, 1 h, 3 h, 4 h, or 6 h later. The rate of Na and Cl loss was greatest during the first 30 min of DH. Net loss of Na and Cl was maximal after 3 h of DH. K loss was slower, achieving significance after 3 h. Electrolyte loss was sufficient to account for observed brain volume regulation after three or more hours of DH. Measurements of /sup 22/Na influx and efflux across the blood brain barrier showed that barrier permeability to Na is unchanged during DH. Analysis of results using a two-compartment model of plasma-brain exchange suggests that loss of brain Na during DH does not result solely from a shift of electrolyte across the blood-brain barrier to plasma, and thus provides indirect evidence for an additional pathway for Na loss, presumably via cerebrospinal fluid.

  20. Procalcitonin behaves as a fast responding acute phase protein in vivo and in vitro

    NARCIS (Netherlands)

    Nijsten, MWN; Olinga, P; The, TH; de Vries, EGE; Groothuis, GMM; Limburg, PC; ten Duis, HJ; Moshage, H; Hoekstra, HJ; Bijzet, J; Zwaveling, JH; Schraffordt Koops, H.

    2000-01-01

    Objectives: Procalcitonin (PCT) is a 13 kD protein of which plasma concentrations are strongly increased in inflammatory states, PCT concentrations are claimed to have a more powerful discriminatory value for bacterial infection than the acute phase proteins serum amyloid A (SAA) or C-reactive prote

  1. E6-Associated Protein Dependent Estrogen Receptor Regulation of Protein Kinase A Regulatory Subunit R2A Expression in Neuroblastoma.

    Science.gov (United States)

    Obeid, Jean-Pierre; Zeidan, Youssef H; Zafar, Nawal; El Hokayem, Jimmy

    2017-02-18

    E6ap is a known transcriptional coregulator for estrogen receptor alpha (Er, Erα) in the presence of estrogen. Protein kinase A (PKA) contains two regulatory subunits derived from four genes. Recent evidence demonstrates that PKA regulates E6ap activity. Data generated in our lab indicated estrogen dependent regulation of Pkar2a levels. Our project sets to investigate a possible feedback mechanism constituting of Erα and E6ap transcriptional regulation of Pkar2a expression. Western blot evaluated protein regulation correlations with E2 in mouse neuroblastoma lines. Bioinformatics detected estrogen response element (ERE) sequences. quantitative polymerase chain reaction (qPCR) validated the western blot results. ERE oligonucleotides were synthesized. Reporter gene transcriptional activity was evaluated via Luciferase assay output. Electromobility shift assay (EMSA) assessed direct binding between Erα relevant sequences. Chromatin immunoprecipitation (ChIP) and Re-ChIP were conducted in quantifying protein complex recruitment levels. Pkar2a protein expression directly correlated with E2, and four putative ERE sequences were identified. Pkar2a mRNA expression reverted to baseline with either E2 or E6ap absent. In the presence of E2, ERE-1 and ERE-4 possessed Luciferase reporter gene transcriptional capabilities. ERE-1 portrayed band shifts, representing direct binding to Erα with E2 supplementation. With E2, ERE-1 significantly enhanced Erα and E6ap recruitment levels to the Pkar2a promoter. Pkar2a is directly regulated by Erα and E6ap in the presence of estrogen stimulus. This work indicates a feedback mechanism in the interplay between PKA and E6ap, which may prove crucial for the role of both proteins in cancers and neurogenetic diseases like Angelman syndrome.

  2. Diagnostical meaning acute phase proteins in cerebrospinal liquid in children with neuroinfections

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    L. A. Alekseeva

    2010-01-01

    Full Text Available In the article presented results of the examination of acute phase proteins in cerebrospinal liquid in 237 children with meningitis and encephalitis viral and bacterial etiology. The dependence between the level of acute phase proteins in cerebrospinal liquid and etiology of neuroinfectional process, the severity of brain damage and the process stage was determined. Diagnostic and prognostic efficiency of the acute phase proteins (C-reactive protein, albumin, alpha-1-antitripsin, alpha-2-macroglobulin, gaptoglobin examination in children with neuroinfections was specified. Developed method of express diagnostics of the severity of inflammatory damage of the brain in bacterial meningitis in children by determination in cerebrospinal liquid alpha-2-macroglobulin is described.

  3. S-100AND#946; protein as a biomarker in acute hemorrhagic stroke

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    Omkar Prasad Baidya

    2014-02-01

    Full Text Available Acute hemorrhagic stroke, a subtype of acute stroke is one of the leading causes of death and disability throughout the world. At present, the diagnosis of acute hemorrhagic stroke is mainly based on Computer Tomography (CT or Magnetic Resonance Imaging (MRI but till now no biomarkers are routinely used in acute hemorrhagic stroke management. This article is a critical and descriptive review on the role of S100β protein as a biomarker in acute hemorrhagic stroke. Plasma S-100β level increases significantly in acute hemorrhagic stroke patients when compared to the normal subjects. Beside, the plasma S-100β can be correlated to the volume of hemorrhage in brain measured by plane CT scan. Plasma S-100β is an useful biomarker in acute hemorrhagic stroke and can be used for estimation of volume of hemorrhage in brain in acute hemorrhagic stroke patients. Thus, S-100β can be useful as an alternative to CT scan/MRI in diagnosis and in taking therapeutic decision in acute hemorrhagic stroke management. [Int J Res Med Sci 2014; 2(1.000: 13-15

  4. Transcriptional control by two leucine-responsive regulatory proteins in Halobacterium salinarum R1

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    Tarasov Valery

    2010-05-01

    Full Text Available Abstract Background Archaea combine bacterial-as well as eukaryotic-like features to regulate cellular processes. Halobacterium salinarum R1 encodes eight leucine-responsive regulatory protein (Lrp-homologues. The function of two of them, Irp (OE3923F and lrpA1 (OE2621R, were analyzed by gene deletion and overexpression, including genome scale impacts using microarrays. Results It was shown that Lrp affects the transcription of multiple target genes, including those encoding enzymes involved in amino acid synthesis, central metabolism, transport processes and other regulators of transcription. In contrast, LrpA1 regulates transcription in a more specific manner. The aspB3 gene, coding for an aspartate transaminase, was repressed by LrpA1 in the presence of L-aspartate. Analytical DNA-affinity chromatography was adapted to high salt, and demonstrated binding of LrpA1 to its own promoter, as well as L-aspartate dependent binding to the aspB3 promoter. Conclusion The gene expression profiles of two archaeal Lrp-homologues report in detail their role in H. salinarum R1. LrpA1 and Lrp show similar functions to those already described in bacteria, but in addition they play a key role in regulatory networks, such as controlling the transcription of other regulators. In a more detailed analysis ligand dependent binding of LrpA1 was demonstrated to its target gene aspB3.

  5. Impairment of Circulating CD4+CD25+GARP+ Regulatory T Cells in Patients with Acute Coronary Syndrome

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    Kai Meng

    2014-02-01

    Full Text Available Background: Atherosclerosis (AS is an inflammatory and immune disease. Regulatory T cells (Tregs suppress the activation of T cells and have been shown to play a protective role during the pathogenesis of AS. However, specific markers for Tregs are lacking. Recently, glycoprotein A repetitions predominant (GARP was discovered as a specific marker of activated Tregs, and we therefore utilized GARP as a specific surface marker for Tregs in the current study. Methods: To assess whether GARP+ Tregs are downregulated in patients with acute coronary syndrome (ACS, we examined CD4+CD25+GARP+ T cell frequencies as well as their associated cytokines and suppressive function. Additionally, we compared GARP expression to that of FOXP3, which may be more sensitive as a marker of activated Tregs in patients with ACS. Results: Patients with ACS demonstrated a significant decrease in circulating CD4+CD25+GARP+ Tregs. Moreover, the suppressive function of Tregs and levels of related cytokines were also impaired in ACS patients compared to those with stable angina (SA or normal coronary artery (NCA. Additionally, after TCR stimulation, peripheral blood mononuclear cells (PBMCs from patients with ACS exhibited a decrease in CD4+CD25+GARP+ Tregs. Conclusions: These fnding indicate that circulating CD4+CD25+GARP+ Tregs are impaired in patients withACS. Thus, targeting GARP may promote the protective function of Tregs in ACS.

  6. Corruption of dendritic cell antigen presentation during acute GVHD leads to regulatory T-cell failure and chronic GVHD.

    Science.gov (United States)

    Leveque-El Mouttie, Lucie; Koyama, Motoko; Le Texier, Laetitia; Markey, Kate A; Cheong, Melody; Kuns, Rachel D; Lineburg, Katie E; Teal, Bianca E; Alexander, Kylie A; Clouston, Andrew D; Blazar, Bruce R; Hill, Geoffrey R; MacDonald, Kelli P A

    2016-08-11

    Chronic graft-versus-host disease (cGVHD) is a major cause of late mortality following allogeneic bone marrow transplantation (BMT) and is characterized by tissue fibrosis manifesting as scleroderma and bronchiolitis obliterans. The development of acute GVHD (aGVHD) is a powerful clinical predictor of subsequent cGVHD, suggesting that aGVHD may invoke the immunologic pathways responsible for cGVHD. In preclinical models in which sclerodermatous cGVHD develops after a preceding period of mild aGVHD, we show that antigen presentation within major histocompatibility complex (MHC) class II of donor dendritic cells (DCs) is markedly impaired early after BMT. This is associated with a failure of regulatory T-cell (Treg) homeostasis and cGVHD. Donor DC-restricted deletion of MHC class II phenocopied this Treg deficiency and cGVHD. Moreover, specific depletion of donor Tregs after BMT also induced cGVHD, whereas adoptive transfer of Tregs ameliorated it. These data demonstrate that the defect in Treg homeostasis seen in cGVHD is a causative lesion and is downstream of defective antigen presentation within MHC class II that is induced by aGVHD.

  7. Aberrant signal transduction and protein expression in acute myeloid leukemia

    NARCIS (Netherlands)

    Schepers, Hein

    2007-01-01

    Het proces van hematopoiese voorziet het lichaam van miljarden bloedcellen per dag. Het is een strak geregisseerd proces. Acute myeloide leukemie (AML) is een afwijking in de bloedcelontwikkeling. Behandeling van deze en andere vormen van leukemie is veelal gebaseerd op het principe van de geprogram

  8. Evolutionary adaptation of an AraC-like regulatory protein in Citrobacter rodentium and Escherichia species.

    Science.gov (United States)

    Tan, Aimee; Petty, Nicola K; Hocking, Dianna; Bennett-Wood, Vicki; Wakefield, Matthew; Praszkier, Judyta; Tauschek, Marija; Yang, Ji; Robins-Browne, Roy

    2015-04-01

    The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogen Citrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and the grlRA operon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genus Escherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated that regA has been horizontally transferred to Escherichia spp. and C. rodentium. Comparative studies of two RegA homologues, namely, those from C. rodentium and E. coli SMS-3-5, a multiresistant environmental strain of E. coli, showed that the two regulators acted similarly in vitro but differed in terms of their abilities to activate the virulence of C. rodentium in vivo, which evidently was due to their differential activation of grlRA. Our data indicate that RegA from C. rodentium has strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence by C. rodentium and on the evolution of virulence-regulatory genes of bacterial pathogens in general.

  9. Signal regulatory protein alpha (SIRPalpha cells in the adaptive response to ESAT-6/CFP-10 protein of tuberculous mycobacteria.

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    W Ray Waters

    Full Text Available BACKGROUND: Early secretory antigenic target-6 (ESAT-6 and culture filtrate protein-10 (CFP-10 are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis involved in phagolysosome escape of the bacillus and, potentially, in the efficient induction of granulomas. Upon tuberculosis infection, multi-nucleate giant cells are elicited, likely as a response aimed at containing mycobacteria. In tissue culture models, signal regulatory protein (SIRPalpha (also referred to as macrophage fusion receptor or CD172a is essential for multi-nucleate giant cell formation. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, ESAT-6/CFP-10 complex and SIRPalpha interactions were evaluated with samples obtained from calves experimentally infected with M. bovis. Peripheral blood CD172a(+ (SIRPalpha-expressing cells from M. bovis-infected calves proliferated upon in vitro stimulation with ESAT-6/CFP-10 (either as a fusion protein or a peptide cocktail, but not with cells from animals receiving M. bovis strains lacking ESAT-6/CFP-10 (i.e, M. bovis BCG or M. bovis DeltaRD1. Sorted CD172a(+ cells from these cultures had a dendritic cell/macrophage morphology, bound fluorescently-tagged rESAT-6:CFP-10, bound and phagocytosed live M. bovis BCG, and co-expressed CD11c, DEC-205, CD44, MHC II, CD80/86 (a subset also co-expressed CD11b or CD8alpha. Intradermal administration of rESAT-6:CFP-10 into tuberculous calves elicited a delayed type hypersensitive response consisting of CD11c(+, CD172a(+, and CD3(+ cells, including CD172a-expressing multi-nucleated giant cells. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate the ability of ESAT-6/CFP-10 to specifically expand CD172a(+ cells, bind to CD172a(+ cells, and induce multi-nucleated giant cells expressing CD172a.

  10. Computational identification of new structured cis-regulatory elements in the 3'-untranslated region of human protein coding genes.

    Science.gov (United States)

    Chen, Xiaowei Sylvia; Brown, Chris M

    2012-10-01

    Messenger ribonucleic acids (RNAs) contain a large number of cis-regulatory RNA elements that function in many types of post-transcriptional regulation. These cis-regulatory elements are often characterized by conserved structures and/or sequences. Although some classes are well known, given the wide range of RNA-interacting proteins in eukaryotes, it is likely that many new classes of cis-regulatory elements are yet to be discovered. An approach to this is to use computational methods that have the advantage of analysing genomic data, particularly comparative data on a large scale. In this study, a set of structural discovery algorithms was applied followed by support vector machine (SVM) classification. We trained a new classification model (CisRNA-SVM) on a set of known structured cis-regulatory elements from 3'-untranslated regions (UTRs) and successfully distinguished these and groups of cis-regulatory elements not been strained on from control genomic and shuffled sequences. The new method outperformed previous methods in classification of cis-regulatory RNA elements. This model was then used to predict new elements from cross-species conserved regions of human 3'-UTRs. Clustering of these elements identified new classes of potential cis-regulatory elements. The model, training and testing sets and novel human predictions are available at: http://mRNA.otago.ac.nz/CisRNA-SVM.

  11. Systematic identification of regulatory proteins critical for T-cell activation

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    Kolbinger Frank

    2003-09-01

    Full Text Available Abstract Background The activation of T cells, mediated by the T-cell receptor (TCR, activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. Results In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 × 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLCγ1 and SHP-1 (PTP1C as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1, transmembrane molecules (EDG1, IL-10Rα and integrin α2, cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1, and cytoskeletal molecules (moesin and vimentin. Furthermore, using truncated Lck, PLCγ1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. Conclusions This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings.

  12. The Multifaceted Activity of the VirF Regulatory Protein in the Shigella Lifestyle

    Science.gov (United States)

    Di Martino, Maria Letizia; Falconi, Maurizio; Micheli, Gioacchino; Colonna, Bianca; Prosseda, Gianni

    2016-01-01

    Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. The highly sophisticated infectious strategy of Shigella banks on the capacity to invade the intestinal epithelial barrier and cause its inflammatory destruction. The cellular pathogenesis and clinical presentation of shigellosis are the sum of the complex action of a large number of bacterial virulence factors mainly located on a large virulence plasmid (pINV). The expression of pINV genes is controlled by multiple environmental stimuli through a regulatory cascade involving proteins and sRNAs encoded by both the pINV and the chromosome. The primary regulator of the virulence phenotype is VirF, a DNA-binding protein belonging to the AraC family of transcriptional regulators. The virF gene, located on the pINV, is expressed only within the host, mainly in response to the temperature transition occurring when the bacterium transits from the outer environment to the intestinal milieu. VirF then acts as anti-H-NS protein and directly activates the icsA and virB genes, triggering the full expression of the invasion program of Shigella. In this review we will focus on the structure of VirF, on its sophisticated regulation, and on its role as major player in the path leading from the non-invasive to the invasive phenotype of Shigella. We will address also the involvement of VirF in mechanisms aimed at withstanding adverse conditions inside the host, indicating that this protein is emerging as a global regulator whose action is not limited to virulence systems. Finally, we will discuss recent observations conferring VirF the potential of a novel antibacterial target for shigellosis. PMID:27747215

  13. The multifaceted activity of the VirF regulatory protein in the Shigella lifestyle

    Directory of Open Access Journals (Sweden)

    Maria Letizia Di Martino

    2016-09-01

    Full Text Available Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. The highly sophisticated infectious strategy of Shigella banks on the capacity to invade the intestinal epithelial barrier and cause its inflammatory destruction. The cellular pathogenesis and clinical presentation of shigellosis are the sum of the complex action of a large number of bacterial virulence factors mainly located on a large virulence plasmid (pINV. The expression of pINV genes is controlled by multiple environmental stimuli through a regulatory cascade involving proteins and sRNAs encoded by both the pINV and the chromosome. The primary regulator of the virulence phenotype is VirF, a DNA-binding protein belonging to the AraC family of transcriptional regulators. The virF gene, located on the pINV, is expressed only within the host, mainly in response to the temperature transition occurring when the bacterium transits from the outer environment to the intestinal milieu. VirF then acts as anti-H-NS protein and directly activates the icsA and virB genes, triggering the full expression of the invasion program of Shigella. In this review we will focus on the structure of VirF, on its sophisticated regulation, and on its role as major player in the path leading from the non invasive to the invasive phenotype of Shigella. We will address also the involvement of VirF in mechanisms aimed at withstanding adverse conditions inside the host, indicating that this protein is emerging as a global regulator whose action is not limited to virulence systems. Finally, we will discuss recent observations conferring VirF the potential of a novel antibacterial target for shigellosis.

  14. Duck hepatitis B virus expresses a regulatory HBx-like protein from a hidden open reading frame.

    Science.gov (United States)

    Chang, S F; Netter, H J; Hildt, E; Schuster, R; Schaefer, S; Hsu, Y C; Rang, A; Will, H

    2001-01-01

    Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thought to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of association of chronic DHBV infection with hepatocellular carcinoma development supports this belief. Here, we demonstrate that DHBV genomes have a hidden open reading frame from which a transcription-regulatory protein, designated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhances neither viral protein expression, intracellular DNA synthesis, nor virion production when assayed in the full-length genome context in LMH cells. However, similar to mammalian hepadnavirus X proteins, DHBx activates cellular and viral promoters via the Raf-mitogen-activated protein kinase signaling pathway and localizes primarily in the cytoplasm. The functional similarities as well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho- and avihepadnavirus X genes. In addition, our data disclose similar intracellular localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinogenesis, and imply unique opportunities for deciphering of their still-enigmatic in vivo functions.

  15. Schiff Base Metal Derivatives Enhance the Expression of HSP70 and Suppress BAX Proteins in Prevention of Acute Gastric Lesion

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    Shahram Golbabapour

    2013-01-01

    Full Text Available Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg, the positive control (Tween 20 5% v/v, 5 mL/kg, and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg. After 1 h, all of the groups received ethanol 95% (5 mL/kg but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg. The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E, immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

  16. Acute phase proteins in bovine milk in an experimental model of Staphylococcus aureus subclinical mastitis

    DEFF Research Database (Denmark)

    Eckersall, P D; Young, F J; Nolan, A M

    2006-01-01

    The objectives were to establish the origin of 2 acute phase proteins in milk during subclinical bovine mastitis and to characterize the relationship between those proteins in milk and blood. Haptoglobin (Hp) and mammary-associated serum amyloid A (M-SAA3) appear in milk during mastitis, whereas Hp...... and serum amyloid A increase in serum during mastitis. The concentrations of these proteins were determined in an experimental model using a field strain of Staphylococcus aureus to induce subclinical mastitis in dairy cows. The expression of mRNA coding for these proteins was assessed and the presence of M......-SAA3 in mammary tissues was determined using immunocytochemistry. Increases of M-SAA3 and Hp in milk occurred within 12 h of Staphylococcus aureus infusion, with peak concentrations occurring 3 d after infusion of the bacteria. The increase of acute phase proteins in milk (15 h) preceded the increase...

  17. Optimal combinations of acute phase proteins for detecting infectious disease in pigs

    DEFF Research Database (Denmark)

    Heegaard, Peter M. H.; Stockmarr, Anders; Piñeiro, Matilde

    2011-01-01

    gondii) and one viral (porcine respiratory and reproductive syndrome virus) infection and one aseptic inflammation. Immunochemical analyses of seven APPs, four positive (C-reactive protein (CRP), haptoglobin (Hp), pig major acute phase protein (pigMAP) and serum amyloid A (SAA)) and three negative......The acute phase protein (APP) response is an early systemic sign of disease, detected as substantial changes in APP serum concentrations and most disease states involving inflammatory reactions give rise to APP responses. To obtain a detailed picture of the general utility of porcine APPs to detect...... for single APPs and for APP combinations. Combinations of APPs allowed the detection of disease more sensitively than any individual APP and the best three-protein combinations were CRP, apoA1, pigMAP and CRP, apoA1, Hp, respectively, closely followed by the two-protein combinations CRP, pigMAP and apoA1...

  18. Hypoxia alters cell cycle regulatory protein expression and induces premature maturation of oligodendrocyte precursor cells.

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    Ravi Shankar Akundi

    Full Text Available BACKGROUND: Periventricular white matter injury (PWMI is a common form of brain injury sustained by preterm infants. A major factor that predisposes to PWMI is hypoxia. Because oligodendrocytes (OLs are responsible for myelination of axons, abnormal OL development or function may affect brain myelination. At present our understanding of the influences of hypoxia on OL development is limited. To examine isolated effects of hypoxia on OLs, we examined the influences of hypoxia on OL development in vitro. METHODOLOGY/FINDINGS: Cultures of oligodendrocyte precursor cells (OPCs were prepared from mixed glial cultures and were 99% pure. OPCs were maintained at 21% O(2 or hypoxia (1% or 4% O(2 for up to 7 days. We observed that 1% O(2 lead to an increase in the proportion of myelin basic protein (MBP-positive OLs after 1 week in culture, and a decrease in the proportion of platelet-derived growth factor receptor alpha (PDGFRalpha-positive cells suggesting premature OL maturation. Increased expression of the cell cycle regulatory proteins p27(Kip1 and phospho-cdc2, which play a role in OL differentiation, was seen as well. CONCLUSIONS: These results show that hypoxia interferes with the normal process of OL differentiation by inducing premature OPC maturation.

  19. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

    Science.gov (United States)

    Garbers, C.; DeLong, A.; Deruere, J.; Bernasconi, P.; Soll, D.; Evans, M. L. (Principal Investigator)

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

  20. Unperturbed posttranscriptional regulatory Rev protein function and HIV-1 replication in astrocytes.

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    Ashok Chauhan

    Full Text Available Astrocytes protect neurons, but also evoke proinflammatory responses to injury and viral infections, including HIV. There is a prevailing notion that HIV-1 Rev protein function in astrocytes is perturbed, leading to restricted viral replication. In earlier studies, our finding of restricted viral entry into astrocytes led us to investigate whether there are any intracellular restrictions, including crippled Rev function, in astrocytes. Despite barely detectable levels of DDX3 (Rev-supporting RNA helicase and TRBP (anti-PKR in primary astrocytes compared to astrocytic cells, Rev function was unperturbed in wild-type, but not DDX3-ablated astrocytes. As in permissive cells, after HIV-1 entry bypass in astrocytes, viral-encoded Tat and Rev proteins had robust regulatory activities, leading to efficient viral replication. Productive HIV-1 infection in astrocytes persisted for several weeks. Our findings on HIV-1 entry bypass in astrocytes demonstrated that the intracellular environment is conducive to viral replication and that Tat and Rev functions are unperturbed.

  1. Dynamic localization of glucokinase and its regulatory protein in hypothalamic tanycytes.

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    Magdiel Salgado

    Full Text Available Glucokinase (GK, the hexokinase involved in glucose sensing in pancreatic β cells, is also expressed in hypothalamic tanycytes, which cover the ventricular walls of the basal hypothalamus and are implicated in an indirect control of neuronal activity by glucose. Previously, we demonstrated that GK was preferentially localized in tanycyte nuclei in euglycemic rats, which has been reported in hepatocytes and is suggestive of the presence of the GK regulatory protein, GKRP. In the present study, GK intracellular localization in hypothalamic and hepatic tissues of the same rats under several glycemic conditions was compared using confocal microscopy and Western blot analysis. In the hypothalamus, increased GK nuclear localization was observed in hyperglycemic conditions; however, it was primarily localized in the cytoplasm in hepatic tissue under the same conditions. Both GK and GKRP were next cloned from primary cultures of tanycytes. Expression of GK by Escherichia coli revealed a functional cooperative protein with a S0.5 of 10 mM. GKRP, expressed in Saccharomyces cerevisiae, inhibited GK activity in vitro with a Ki 0.2 µM. We also demonstrated increased nuclear reactivity of both GK and GKRP in response to high glucose concentrations in tanycyte cultures. These data were confirmed using Western blot analysis of nuclear extracts. Results indicate that GK undergoes short-term regulation by nuclear compartmentalization. Thus, in tanycytes, GK can act as a molecular switch to arrest cellular responses to increased glucose.

  2. Regional protein synthesis in rat brain following acute hemispheric ischemia.

    Science.gov (United States)

    Dienel, G A; Pulsinelli, W A; Duffy, T E

    1980-11-01

    Regional protein synthesis was measured in rat brain at intervals up to 48 h following occlusion of the four major arteries to the brain for either 10 or 30 min. Four-vessel occlusions produces ischemia in the cerebral hemispheres and oligemia in the midbrain-diencephalon and brainstem. During the hour following 10 min of ischemia, protein synthesis, measured by incorporation of [14C]valine into protein, was inhibited in the cerebral cortex by 67%. Normal rates of protein synthesis were attained within 4 h of recirculation. In rats subjected to 30 min of ischemia, protein synthesis was inhibited by 83% during the first hour of recirculation in the cortex, caudate-putamen, and hippocampus. Recovery of protein synthesis in these regions was slow (25-48 h). The midbrain-diencephalon showed less inhibition, 67%, and faster recovery (by 12 h). Protein synthesis was unaffected in the brainstem. [14C]Autoradiography revealed that the pyramidal neurons of the hippocampus and areas of the caudate and cortex failed to recover normal rates of protein synthesis even after 48 h. The accumulation of TCA-soluble [14C]valine was enhanced (55-65%) in the cortex, caudate, and hippocampus after 30 min of ischemia; the increase persisted for 12 h. A smaller rise in [14C]valine content (30%) and more rapid normalization of valine accumulation (by 7 h) were observed in the midbrain-diencephalon; no changes were found in the brainstem. In the cortex, recovery was more rapid when the duration of ischemia was reduced. Thus, the degree of inhibition of protein synthesis, the accumulation of valine in the tissue, and the length of time required to reestablish normal values for these processes were dependent on both the severity and the duration of the ischemic insult. Restoration of normal rates of protein synthesis after ischemia was slow compared with the normalization of cerebral energy metabolites.

  3. Innate immunity and protective neuroinflammation: new emphasis on the role of neuroimmune regulatory proteins.

    Science.gov (United States)

    Griffiths, M; Neal, J W; Gasque, P

    2007-01-01

    Brain inflammation due to infection, hemorrhage, and aging is associated with activation of the local innate immune system as expressed by infiltrating cells, resident glial cells, and neurons. The innate immune response relies on the detection of "nonself" and "danger-self" ligands behaving as "eat me signals" by a plethora of pattern recognition receptors (PRRs) expressed by professional and amateur phagocytes to promote the clearance of pathogens, toxic cell debris (amyloid fibrils, aggregated synucleins, prions), and apoptotic cells accumulating within the brain parenchyma and the cerebrospinal fluid (CSF). These PRRs (e.g., complement, TLR, CD14, scavenger receptors) are highly conserved between vertebrates and invertebrates and may represent the most ancestral innate scavenging system involved in tissue homeostasis. However, in some diseases, these protective mechanisms lead to neurodegeneration on the ground that several innate immune molecules have neurocytotoxic activities. The response is a "double-edged sword" representing a fine balance between protective and detrimental effects. Several key regulatory mechanisms have now been evidenced in the control of CNS innate immunity, and these could be harnessed to explore novel therapeutic avenues. We will herein provide new emphasis on the role of neuroimmune regulatory proteins (NIRegs), such as CD95L, TNF, CD200, CD47, sialic acids, CD55, CD46, fH, C3a, HMGB1, which are involved in silencing innate immunity at the cellular and molecular levels and suppression of inflammation. For instance, NIRegs may play an important role in controlling lymphocyte/macrophage/microglia hyperinflammatory responses, while sparing host defense and repair mechanisms. Moreover, NIRegs have direct beneficial effects on neurogenesis and contributing to brain tissue remodeling.

  4. Alterations of intestinal immune function and regulatory effects of L-arginine in experimental severe acute pancreatitis rats

    Institute of Scientific and Technical Information of China (English)

    Shi-Feng Qiao; Tian-Jing Lü; Jia-Bang Sun; Fei Li

    2005-01-01

    AIM: To discuss the changes of intestinal mucosal immune function in rats with experimental severe acute pancreatitis(SAP) and the regulatory effect of L-arginine.METHODS: Male adult Wistar rats were randomly divided into pancreatitis group, sham-operation group, and L-arginine treatment group. Animals were killed at 24, 48, and 72 h after SAP models were developed and specimens were harvested. Endotoxin concentration in portal vein was determined by limulus endotoxin analysis kit. CD3+, CD4+,CD8+ T lymphocytes in intestinal mucosal lamina propria were examined by immunohistochemistry. Secretory immunoglobulin A (SIgA) in cecum feces was examined by radioimmunoassay.RESULTS: Compared to the control group, plasma endotoxin concentration in the portal vein increased, percentage of CD3+ and CD4+ T lymphocyte subsets in the end of intestinal mucosal lamina propria reduced significantly,CD4+/CD8+ ratio decreased, and SIgA concentrations in cecum feces reduced at 24, 48, and 72 h after SAP developed. Compared to SAP group, the L-arginine treatment group had a lower level of plasma endotoxin concentration in the portal vein, a higher CD3+ and CD4+ T lymphocyte percentage in the end of intestinal mucosal lamina propria,an increased ratio of CD4+/CD8+ and a higher SIgA concentration in cecum feces.CONCLUSION: Intestinal immune suppression occurs in the early stage of SAP rats, which may be the main reason for bacterial and endotoxin translocation. L-arginine can improve the intestinal immunity and reduce bacterial and endotoxin translocation in SAP rats.

  5. Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

    Science.gov (United States)

    Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

    2015-02-01

    sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo.

  6. Acute supplementation of amino acids increases net protein accretion in IUGR fetal sheep.

    Science.gov (United States)

    Brown, Laura D; Rozance, Paul J; Thorn, Stephanie R; Friedman, Jacob E; Hay, William W

    2012-08-01

    Placental insufficiency decreases fetal amino acid uptake from the placenta, plasma insulin concentrations, and protein accretion, thus compromising normal fetal growth trajectory. We tested whether acute supplementation of amino acids or insulin into the fetus with intrauterine growth restriction (IUGR) would increase net fetal protein accretion rates. Late-gestation IUGR and control (CON) fetal sheep received acute, 3-h infusions of amino acids (with euinsulinemia), insulin (with euglycemia and euaminoacidemia), or saline. Fetal leucine metabolism was measured under steady-state conditions followed by a fetal muscle biopsy to quantify insulin signaling. In CON, increasing amino acid delivery rates to the fetus by 100% increased leucine oxidation rates by 100%. In IUGR, amino acid infusion completely suppressed fetal protein breakdown rates but increased leucine oxidation rate by only 25%, resulting in increased protein accretion rates by 150%. Acute insulin infusion, however, had very little effect on amino acid delivery rates, fetal leucine disposal rates, or fetal protein accretion rates in CON or IUGR fetuses despite robust signaling of the fetal skeletal muscle insulin-signaling cascade. These results indicate that, when amino acids are given directly into the fetal circulation independently of changes in insulin concentrations, IUGR fetal sheep have suppressed protein breakdown rates, thus increasing net fetal protein accretion.

  7. Phage phi 29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein-protein contacts.

    Science.gov (United States)

    Nuez, B; Rojo, F; Salas, M

    1992-12-01

    Transcription from the late promoter, PA3, of Bacillus subtilis phage phi 29 is activated by the viral regulatory protein p4. A kinetic analysis of the activation process has revealed that the role of protein p4 is to stabilize the binding of RNA polymerase to the promoter as a closed complex without significantly affecting further steps of the initiation process. Electrophoretic band-shift assays performed with a DNA fragment spanning only the protein p4 binding site showed that RNA polymerase could efficiently retard the complex formed by protein p4 bound to the DNA. Similarly, when a DNA fragment containing only the RNA polymerase-binding region of PA3 was used, p4 greatly stimulated the binding of RNA polymerase to the DNA. These results strongly suggest that p4 and RNA polymerase contact each other at the PA3 promoter. In the light of current knowledge of the p4 activation mechanism, we propose that direct contacts between the two proteins participate in the activation process.

  8. The porcine acute phase protein response to acute clinical and subclinical experimental infection with Streptococcus suis

    DEFF Research Database (Denmark)

    Sørensen, Nanna Skall; Tegtmeier, C.; Andresen, Lars Ole;

    2006-01-01

    and apolipoprotein (Apo) A-I. The aim was to elucidate the differences in the acute phase behaviour of the individual APPs during a typical bacterial septicaemic, infection. Pigs were inoculated subcutaneously with live S. suis serotype 2 and blood was sampled before and on various days post inoculation (p...... the experiment with maximum levels around 10 times the day 0-levels, and pig-MAP was elevated on days 1-12 p.i. with peak levels of around seven times the day 0-levels. Apo A-I was decreased from days 1 to 8 and showed minimum levels of about 40% of day 0-levels around 1-2 days p.i. No clear pattern of changes...... signs and no arthritic lesions showed an APP response comparable to that of the other, clinically affected pigs. Thus, both acute clinical and subclinical S. suis infection could be revealed by the measurement of one or more of the APPs CRP, SAA, Hp, pig-MAP and Apo A-I. The combined measurement of two...

  9. Identification of Functional Regulatory Residues of the β-Lactam Inducible Penicillin Binding Protein in Methicillin-Resistant Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Andreas N. Mbah

    2013-01-01

    Full Text Available Resistance to methicillin by Staphylococcus aureus is a persistent clinical problem worldwide. A mechanism for resistance has been proposed in which methicillin resistant Staphylococcus aureus (MRSA isolates acquired a new protein called β-lactam inducible penicillin binding protein (PBP-2′. The PBP-2′ functions by substituting other penicillin binding proteins which have been inhibited by β-lactam antibiotics. Presently, there is no structural and regulatory information on PBP-2′ protein. We conducted a complete structural and functional regulatory analysis of PBP-2′ protein. Our analysis revealed that the PBP-2′ is very stable with more hydrophilic amino acids expressing antigenic sites. PBP-2′ has three striking regulatory points constituted by first penicillin binding site at Ser25, second penicillin binding site at Ser405, and finally a single metallic ligand binding site at Glu657 which binds to Zn2+ ions. This report highlights structural features of PBP-2′ that can serve as targets for developing new chemotherapeutic agents and conducting site direct mutagenesis experiments.

  10. Identification of Functional Regulatory Residues of the β -Lactam Inducible Penicillin Binding Protein in Methicillin-Resistant Staphylococcus aureus.

    Science.gov (United States)

    Mbah, Andreas N; Isokpehi, Raphael D

    2013-01-01

    Resistance to methicillin by Staphylococcus aureus is a persistent clinical problem worldwide. A mechanism for resistance has been proposed in which methicillin resistant Staphylococcus aureus (MRSA) isolates acquired a new protein called β -lactam inducible penicillin binding protein (PBP-2'). The PBP-2' functions by substituting other penicillin binding proteins which have been inhibited by β -lactam antibiotics. Presently, there is no structural and regulatory information on PBP-2' protein. We conducted a complete structural and functional regulatory analysis of PBP-2' protein. Our analysis revealed that the PBP-2' is very stable with more hydrophilic amino acids expressing antigenic sites. PBP-2' has three striking regulatory points constituted by first penicillin binding site at Ser25, second penicillin binding site at Ser405, and finally a single metallic ligand binding site at Glu657 which binds to Zn(2+) ions. This report highlights structural features of PBP-2' that can serve as targets for developing new chemotherapeutic agents and conducting site direct mutagenesis experiments.

  11. The effect of chronic ammonia exposure on acute phase proteins, immunoglobulin and cytokines in laying hens

    Science.gov (United States)

    Ammonia is a potential health hazard to both humans and animals, causing systemic low-grade inflammation based on its levels and durations. The objective of this study was to examine the effect of 45 weeks of exposure to 30 ppm NH3 on the concentrations of acute phase proteins, immunoglobulins and c...

  12. Expression of multidrug resistance-associated proteins predicts prognosis in childhood and adult acute lymphoblastic leukemia

    NARCIS (Netherlands)

    Plasschaert, SLA; de Bont, ESJM; Boezen, M; vander Kolk, DM; Daenen, SMJG; Faber, KN; Kamps, WA; de Vries, EGE; Vellenga, E

    2005-01-01

    PURPOSE: Patients with acute lymphoblastic leukemia (ALL) are treated with a variety of chemotherapeutic drugs, which can be transported by six multidrug resistance-associated proteins (MRP). These MRPs have strongly overlapping functional activities. The aim of this study was to investigate the exp

  13. PLASMA PROTEIN ELECTROPHORESIS AND SELECT ACUTE PHASE PROTEINS IN HEALTHY BONNETHEAD SHARKS (SPHYRNA TIBURO) UNDER MANAGED CARE.

    Science.gov (United States)

    Hyatt, Michael W; Field, Cara L; Clauss, Tonya M; Arheart, Kristopher L; Cray, Carolyn

    2016-12-01

    Preventative health care of elasmobranchs is an important but understudied field of aquatic veterinary medicine. Evaluation of inflammation through the acute phase response is a valuable tool in health assessments. To better assess the health of bonnethead sharks ( Sphyrna tiburo ) under managed care, normal reference intervals of protein electrophoresis (EPH) and the acute phase proteins, C-reactive protein (CRP) and haptoglobin (HP), were established. Blood was collected from wild caught, captive raised bonnethead sharks housed at public aquaria. Lithium heparinized plasma was either submitted fresh or stored at -80°C prior to submission. Electrophoresis identified protein fractions with migration characteristics similar to other animals with albumin, α-1 globulin, α-2 globulin, β globulin, and γ globulin. These fractions were classified as fractions 1-5 as fractional contents are unknown in this species. Commercial reagents for CRP and HP were validated for use in bonnethead sharks. Reference intervals were established using the robust method recommended by the American Society for Veterinary Clinical Pathology for the calculation of 90% reference intervals. Once established, the diagnostic and clinical applicability of these reference intervals was used to assess blood from individuals with known infectious diseases that resulted in systemic inflammation and eventual death. Unhealthy bonnethead sharks had significantly decreased fraction 2, fraction 3, and fraction 3:4 ratio and significantly increased fraction 5, CRP, and HP. These findings advance our understanding of elasmobranch acute phase inflammatory response and health and aid clinicians in the diagnosis of inflammatory disease in bonnethead sharks.

  14. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  15. Importance of fumarate and nitrate reduction regulatory protein for intestinal proliferation of Vibrio vulnificus.

    Science.gov (United States)

    Kado, Takehiro; Kashimoto, Takashige; Yamazaki, Kohei; Ueno, Shunji

    2017-01-01

    The sepsis caused by Vibrio vulnificus is characterized by an average incubation period of 26 h and a high mortality rate exceeding 50%. The fast growth and dissemination of V. vulnificus in vivo lead to poor clinical outcomes in patients. Therefore, elucidation of the proliferation mechanisms of this organism in vivo may lead to the development of an effective therapeutic strategy. In this study, we focused on the low oxygen concentration in the intestinal milieu because of its drastic difference from that in air. Fumarate and nitrate reduction regulatory protein (FNR) is known to be a global transcriptional regulator for adaptation to anaerobic conditions in various bacteria. We generated a strain of V. vulnificus in which the fnr gene was replaced with an erythromycin resistance gene (fnr::erm mutant). When the fnr::erm mutant was tested in a growth competition assay against the wild-type (WT) in vivo, the competitive index of fnr::erm mutant to WT in the intestinal loop and liver was 0.378 ± 0.192 (mean ± SD) and 0.243 ± 0.123, respectively. These data suggested that FNR is important for the proliferation of V. vulnificus in the intestine to achieve a critical mass to be able to invade the systemic circulation.

  16. Crystal structures of catalytic and regulatory subunits of rat protein kinase CK2

    Institute of Scientific and Technical Information of China (English)

    ZHOU WeiHong; SHEN YueQuan; QIN XiaoHong; YAN XiaoJie; XIE XingQiao; LI Liang; FANG ShaSha; LONG JiaFu; ADELMAN John; TANG Wei-Jen

    2009-01-01

    Protein kinase CK2 consists of two catalytic subunits (CK2α) and two regulatory subunits (CK2β). Here,we report the crystal structures of rat CK2α mutant (rCK2α-△C, 1-335) and CK2β(rCK2β). The overall topology of rCK2α-△C and rCK2βare very similar to the human enzyme, although large structural dif-ferences could be observed in the N-terminal domain of rCK2α-△C. Our reported structure of rCK2α-△C is in the close conformation state while the counterpart hCK2α is in the open conformation state, indi-cating the conformation of CK2α molecule has high plasticity. The structure of rCK2β represents the conformation of free CK2β. Upon CK2α binding, the C-terminal region undergoes a drastic conforma-tional change. The major region of interaction within the interface of CK2αCK2β may serve as a bridge to transmit the conformational change and thus regulate the activity of CK2α.

  17. Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

    Directory of Open Access Journals (Sweden)

    Andreas Bitter

    2015-01-01

    Full Text Available Background/Aims: Sterol regulatory element-binding protein (SREBP 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specifc and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specifc knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression.

  18. Heat Shock Protein 90 Modulates Lipid Homeostasis by Regulating the Stability and Function of Sterol Regulatory Element-binding Protein (SREBP) and SREBP Cleavage-activating Protein.

    Science.gov (United States)

    Kuan, Yen-Chou; Hashidume, Tsutomu; Shibata, Takahiro; Uchida, Koji; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2017-02-17

    Sterol regulatory element-binding proteins (SREBPs) are the key transcription factors that modulate lipid biosynthesis. SREBPs are synthesized as endoplasmic reticulum-bound precursors that require proteolytic activation in the Golgi apparatus. The stability and maturation of precursor SREBPs depend on their binding to SREBP cleavage-activating protein (SCAP), which escorts the SCAP-SREBP complex to the Golgi apparatus. In this study, we identified heat shock protein (HSP) 90 as a novel SREBP regulator that binds to and stabilizes SCAP-SREBP. In HepG2 cells, HSP90 inhibition led to proteasome-dependent degradation of SCAP-SREBP, which resulted in the down-regulation of SREBP target genes and the reduction in intracellular triglyceride and cholesterol levels. We also demonstrated in vivo that HSP90 inhibition decreased SCAP-SREBP protein, down-regulated SREBP target genes, and reduced lipids levels in mouse livers. We propose that HSP90 plays an indispensable role in SREBP regulation by stabilizing the SCAP-SREBP complex, facilitating the activation of SREBP to maintain lipids homeostasis.

  19. 电磁辐射后大鼠睾丸类固醇激素合成急性调节蛋白和细胞色素P450胆固醇侧链裂解酶mRNA的变化%Change of steroidogenic acute regulatory protein and cytochrome P450 cholesterol side-chain cleavage mRNA in testis of rats after electromagnetic irradiation

    Institute of Scientific and Technical Information of China (English)

    周文; 杨进清; 王虚步; 刘勇; 张广斌

    2005-01-01

    electromagnetic irradiation.But under the condition of electromagnetic exposure,the change of testosterone and its mechanism remain unknown. OBJECTIVE:To study the effect of electromagnetic irradiation on steroidogenic acute regulatory protein(StAR) and cytochrome P450 cholesterol side-chain cleavage(P450scc) in the tissues of the testis of the adult rats,and to preliminarily reveal the molecular mechanism of electromagnetic radiation that affects the synthesis of testosterone. DESIGN:A randomized controlled study. SETTING:Department of Nuclear Medicine and Staff Room of Labour Hygiene, Institute of Field Surgery,Daping Hospital of the Third Military Medical University of Chinese PLA. PARTICIPANTS:The study was completed in the Department of Nuclear Medicine and Staff Room of Labour Hygiene, Institute of Field Surgery,Daping Hospital of the Third Military Medical University of Chinese PLA from June 2003 to May 2004.A total of 75 male Wistar rats of clean degree II,weighing 180 to 220 g,were randomly divided into 5 groups:control group,and groups of 3,6,24,72 hours after electromagnetic irradiation with 15 rats in each group. INTERVENTIONS:The rats were exposed to microwave with the peak power of 90 W/cm2 for 15 minutes successively,and all the rats survived the irradiation.The serum contents of testosterone of rats in the groups of 3,6,24,72 hours after electromagnetic irradiation and control group were measured by using radioimmunoassay(RIA) respectively;meanwhile the levels of the StAR and P450scc mRNA in testis tissue were also determined with RT-PCR. MAIN OUTCOME MEASURES:Serum contents of testosterone in the groups of 3,6,24,72 hours after electromagnetic irradiation and control group;the expression levels of the StAR and P450scc mRNA in testis tissues. RESULTS:The serum contents of testosterone,the StAR and P450scc mRNA levels at the point of 3 hours after electromagnetic irradiation were significantly lower than those in the control group,and they were decreased by 83

  20. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE of urinary protein in acute kidney injury

    Directory of Open Access Journals (Sweden)

    Sufi M Suhail

    2011-01-01

    Full Text Available Recent experimental and clinical studies have shown the importance of urinary proteomics in acute kidney injury (AKI. We analyzed the protein in urine of patients with clinical AKI using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE for its diagnostic value, and followed them up for 40 months to evaluate prognosis. Urine from 31 consecutive cases of AKI was analyzed with SDS-PAGE to determine the low, middle and high molecular weight proteins. Fractional excretion of sodium (FENa was estimated from serum and urine creatinine and sodium (Na. The cases were followed-up for 40 months from the end of the recruitment of study cases. Glomerular protein was higher in the hematuria group when compared with the non-hematuria group (P <0.04 and in the AKI group than in the acute on chronic renal failure (AKI-on-CRF group (P <0.002. Tubular protein was higher in the AKI-on-CRF group (P <0.003 than in the AKI group. Tubular protein correlated with FENa in groups with diabetes mellitus (DM, AKI-on-CRF, and without hematuria (P <0.03, P <0.02 and P <0.004, respectively. Pattern of protein did not differ between groups with and without DM and clinical acute tubular necrosis (ATN. At the end of 40 months follow-up, category with predominantly glomerular protein progressed to chronic renal failure (CRF or end-stage renal failure in higher proportion (P <0.05. In clinical AKI, we observed that glomerular protein dominated in cases with glomerular insult, as indicated by hematuria. Tubular protein was common in the study cases with CRF, DM and cases without hematuria. This indicates tubulo-interstitial injury for AKI in these cases. Patients with predominantly glomerular protein had an adverse outcome.

  1. Assessment of heat shock proteins and endothelial dysfunction in acute pulmonary embolism.

    Science.gov (United States)

    İn, Erdal; Deveci, Figen; Kaman, Dilara

    2016-06-01

    We determined the levels of some heat shock proteins (HSP27, HSP70, and HSP90), L-arginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) levels in patients with acute pulmonary embolism. The present case-control study comprised a healthy control group (n = 57) and patients with acute pulmonary embolism (n = 84). HSPs, L-arginine, ADMA, and SDMA levels were measured in all of the cases. The mean age of the control group was 56.72 ± 8.44 years, and the mean age of the patients with acute pulmonary embolism was 60.20 ± 16.56 years (P = 0.104). Compared with controls, patients with acute pulmonary embolism had significantly higher mean serum HSP27, HSP90, and ADMA levels, whereas the mean serum L-arginine and SDMA levels were lower (P In patients with acute pulmonary embolism serum HSP27, HSP70, and ADMA levels were negatively correlated with partial pressures of arterial oxygen levels (r = -0.281, P = 0.01; r = -0.263, P = 0.016; and r = -0.275, P = 0.011, respectively) and arterial oxygen saturation (r = -0.225, P = 0.039; r = -0.400, P in acute pulmonary embolism.

  2. Circulating microbial products and acute phase proteins as markers of pathogenesis in lymphatic filarial disease.

    Directory of Open Access Journals (Sweden)

    R Anuradha

    Full Text Available Lymphatic filariasis can be associated with development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Dysregulated host inflammatory responses leading to systemic immune activation are thought to play a central role in filarial disease pathogenesis. We measured the plasma levels of microbial translocation markers, acute phase proteins, and inflammatory cytokines in individuals with chronic filarial pathology with (CP Ag+ or without (CP Ag- active infection; with clinically asymptomatic infections (INF; and in those without infection (endemic normal [EN]. Comparisons between the two actively infected groups (CP Ag+ compared to INF and those without active infection (CP Ag- compared to EN were used preliminarily to identify markers of pathogenesis. Thereafter, we tested for group effects among all the four groups using linear models on the log transformed responses of the markers. Our data suggest that circulating levels of microbial translocation products (lipopolysaccharide and LPS-binding protein, acute phase proteins (haptoglobin and serum amyloid protein-A, and inflammatory cytokines (IL-1β, IL-12, and TNF-α are associated with pathogenesis of disease in lymphatic filarial infection and implicate an important role for circulating microbial products and acute phase proteins.

  3. Age- and Sex-Associated Effects on Acute-Phase Proteins in Göttingen Minipigs

    DEFF Research Database (Denmark)

    Christoffersen, Berit Ø; Jensen, Søren J.; Ludvigsen, Trine P

    2015-01-01

    Göttingen minipigs are a useful model for diseases having an inflammatory component, and the associated use of acute-phase proteins (APP) as biomarkers of inflammation warrants establishment of their reference ranges. The objective of this study was to establish reference values for selected APP...... in Göttingen minipigs and to investigate the effects of age, sex, and various stimuli on these ranges. Serum concentrations of C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin, pig major acute-phase protein (PMAP), albumin, and porcine α-1 acid glycoprotein (PAGP) were evaluated in 4 age groups (6...... in obese pigs without diabetes. In conclusion, reference values for CRP, PMAP, haptoglobin, SAA, PAGP and albumin were established for male and female Göttingen minipigs of different ages. These APP were influenced by age and sex, underlining the importance of considering these factors when designing...

  4. Usefulness of pregnancy-associated plasma protein A in patients with acute coronary syndrome

    DEFF Research Database (Denmark)

    Iversen, Kasper; Dalsgaard, Morten; Teisner, Ane S

    2009-01-01

    To investigate whether pregnancy-associated plasma protein-A (PAPP-A) is a prognostic marker in patients admitted with high-risk acute coronary syndrome. In patients admitted with high-risk non-ST-segment elevation acute coronary syndrome (NSTE-ACS) and ST-segment elevation myocardial infarction...... (STEMI), risk stratification is primarily determined by the markers of myocardial necrosis and known demographic risk profiles. However, it has recently been proposed that the presence and extent of vulnerable plaques might influence the prognosis significantly. A marker for the vulnerable plaque could...

  5. Role of CC chemokines (macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, RANTES) in acute lung injury in rats

    DEFF Research Database (Denmark)

    Bless, N M; Huber-Lang, M; Guo, R F

    2000-01-01

    were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1 beta and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response...... that in chemokine-dependent inflammatory responses in lung CC chemokines do not necessarily demonstrate redundant function.......The role of the CC chemokines, macrophage inflammatory protein-1 beta (MIP-1 beta), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1 beta, MCP-1, and RANTES...

  6. Source of dietary protein influences kinetics of plasma gut regulatory peptide concentration in response to feeding in preruminant calves.

    Science.gov (United States)

    le Huërou-Luron, I; Gestin, M; Le Dréan, G; Romé, V; Bernard, C; Chayvialle, J A; Guilloteau, P

    1998-03-01

    The kinetics of the peripheral plasma concentrations of eight gut regulatory peptides were examined in response to feeding in preruminant calves. Two experiments were carried out in animals fed milk substitutes either based on milk protein (control diet) or in which casein had been replaced by hydrolyzed fish (fish diet in experiment 1) or whey (whey diet in experiment 2) protein concentrate. In contrast to the control diet, the latter two did not coagulate within the abomasum. No variation was observed in plasma concentrations of gut regulatory peptides during 1-1.4 hr before the morning meal regardless of the nature of the dietary protein. With the control diet, the meal was followed by an increase in cholecystokinin, gastrin and gastric inhibitory polypeptide and a fall in secretin, vasoactive intestinal polypeptide and motilin, whereas no significant change was observed for somatostatin and pancreatic polypeptide. The replacement of casein by protein substitutes did not greatly modify the pattern of plasma responses to feeding, but the prefeeding and postfeeding levels were highly affected. We conclude that the most important characteristic influencing plasma gut peptide concentrations is the ability of dietary protein to clot in the abomasum, consequently determining the pattern of gastric emptying, and that variations appear depending on the origin of protein substitutes in relation to the duodenal content and mainly to the digesta pH.

  7. The ratio of circulating regulatory T cells (Tregs)/Th17 cells is associated with acute allograft rejection in liver transplantation.

    Science.gov (United States)

    Wang, Ying; Zhang, Min; Liu, Zhen-Wen; Ren, Wei-Guo; Shi, Yan-Chao; Sun, Yan-Ling; Wang, Hong-Bo; Jin, Lei; Wang, Fu-Sheng; Shi, Ming

    2014-01-01

    CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) and Th17 cells are known to be involved in the alloreactive responses in organ transplantation, but little is known about the relationship between Tregs and Th17 cells in the context of liver alloresponse. Here, we investigated whether the circulating Tregs/Th17 ratio is associated with acute allograft rejection in liver transplantation. In present study, thirty-eight patients who received liver transplant were enrolled. The patients were divided into two groups: acute allograft rejection group (Gr-AR) (n = 16) and stable allograft liver function group (Gr-SF) (n = 22). The frequencies of circulating Tregs and circulating Th17 cells, as well as Tregs/Th17 ratio were determined using flow cytometry. The association between Tregs/Th17 ratio and acute allograft rejection was then analyzed. Our results showed that the frequency of circulating Tregs was significantly decreased, whereas the frequency of circulating Th17 cells was significantly increased in liver allograft recipients who developed acute rejection. Tregs/Th17 ratio had a negative correlation with liver damage indices and the score of rejection activity index (RAI) after liver transplantation. In addition, the percentages of CTLA-4(+), HLA-DR(+), Ki67(+), and IL-10(+) Tregs were higher in Gr-SF group than in Gr-AR group. Our results suggested that the ratio of circulating Tregs/Th17 cells is associated with acute allograft rejection, thus the ratio may serve as an alternative marker for the diagnosis of acute rejection.

  8. Influence of oxygen on DNA binding, positive control, and stability of the Bradyrhizobium japonicum NifA regulatory protein.

    OpenAIRE

    1991-01-01

    Central to the genetic regulatory circuit that controls Bradyrhizobium japonicum nif and fix gene expression is the NifA protein. NifA activates transcription of several nif and fix genes and autoregulates its expression during symbiosis in soybean root nodules or in free-living microaerobic conditions. High O2 tensions result in the lack of nif expression, possibly by inactivation of NifA through oxidation of an essential metal cofactor. Several B. japonicum nif and fix promoters have upstre...

  9. The complement regulatory protein CD59: insights into attenuation of choroidal neovascularization.

    Science.gov (United States)

    Schnabolk, Gloriane; Tomlinson, Stephen; Rohrer, Bärbel

    2014-01-01

    Complement activation is associated with age-related macular degeneration (AMD), with the retinal pigment epithelium (RPE) being one of the main target tissues. In AMD, disease severity is correlated with the formation of the membrane attack complex (MAC), the terminal step in the complement cascade, as well as diminished RPE expression of CD59, a membrane-bound regulatory protein of MAC formation. This has prompted the search for therapeutic strategies based on MAC inhibition, and soluble forms of CD59 (sCD59) have been investigated in mouse laser-induced choroidal neovascularization, a model for "wet" AMD. Unlike membrane-bound CD59, sCD59 provides relatively poor cell protection from complement, and different strategies to increase sCD59 activity at the cell membrane level have been investigated. These include increasing the circulatory half-life of sCD59 by the addition of an Fc moiety; increasing the half-life of sCD59 in target tissues by modifying CD59 with a (non-specific) membrane-targeting domain; and by locally overexpressing sCD59 via adenoviral vectors. Finally, a different strategy currently under investigation employs complement receptor (CR)2-mediated targeting of CD59 exclusively to membranes under complement attack. CR2 recognizes long-lasting membrane-bound breakdown activation fragments of complement C3. CR2-CD59 may have greater therapeutic potential than other complement inhibitory approaches, since it can be administered either systemically or locally, it will bind specifically to membranes containing activated complement activation fragments, and dosing can be regulated. Hence, this strategy might offer opportunities for site-specific inhibition of complement in diseases with restricted sites of inflammation such as AMD.

  10. Acute-phase protein α1-antitrypsin--a novel regulator of angiopoietin-like protein 4 transcription and secretion.

    Science.gov (United States)

    Frenzel, Eileen; Wrenger, Sabine; Immenschuh, Stephan; Koczulla, Rembert; Mahadeva, Ravi; Deeg, H Joachim; Dinarello, Charles A; Welte, Tobias; Marcondes, A Mario Q; Janciauskiene, Sabina

    2014-06-01

    The angiopoietin-like protein 4 (angptl4, also known as peroxisome proliferator-activated receptor [PPAR]γ-induced angiopoietin-related protein) is a multifunctional protein associated with acute-phase response. The mechanisms accounting for the increase in angptl4 expression are largely unknown. This study shows that human α1-antitrypsin (A1AT) upregulates expression and release of angplt4 in human blood adherent mononuclear cells and in primary human lung microvascular endothelial cells in a concentration- and time-dependent manner. Mononuclear cells treated for 1 h with A1AT (from 0.1 to 4 mg/ml) increased mRNA of angptl4 from 2- to 174-fold, respectively, relative to controls. In endothelial cells, the maximal effect on angptl4 expression was achieved at 8 h with 2 mg/ml A1AT (11-fold induction versus controls). In 10 emphysema patients receiving A1AT therapy (Prolastin), plasma angptl4 levels were higher relative to patients without therapy (nanograms per milliliter, mean [95% confidence interval] 127.1 [99.5-154.6] versus 76.8 [54.8-98.8], respectively, p = 0.045) and correlated with A1AT levels. The effect of A1AT on angptl4 expression was significantly diminished in cells pretreated with a specific inhibitor of ERK1/2 activation (UO126), irreversible and selective PPARγ antagonist (GW9662), or genistein, a ligand for PPARγ. GW9662 did not alter the ability of A1AT to induce ERK1/2 phosphorylation, suggesting that PPARγ is a critical mediator in the A1AT-driven angptl4 expression. In contrast, the forced accumulation of HIF-1α, an upregulator of angptl4 expression, enhanced the effect of A1AT. Thus, acute-phase protein A1AT is a physiological regulator of angptl4, another acute-phase protein.

  11. Acute phase proteins as diagnostic markers in horses with colic

    DEFF Research Database (Denmark)

    Pihl, Tina Holberg; Scheepers, Elrien; Sanz, Macarena

    and fibrinogen to differentiate between horses with infectious non-surgical colic and surgical colic. Materials & Methods:The performance of the APPs was evaluated individually and in combination with clinical examination, as wells as traditional biomarkers in blood (WCC, PCV, TPP, lactate) and peritoneal fluid...... (PF) (haemolysis, WCC, total protein).Admission data collected prospectively from 148 horses with severe colic in one hospital was used to construct multivariate logistic models to predict if a horse had an infectious non-surgical colic. The models were based on 1) clinical evaluation, 2) clinical...... and blood evaluation and 3) clinical, blood and PF evaluation. Each model was independently validated against admission data from 78 horses in another hospital.Results and Discussion:The variables included in the final ‘clinical model’ were: Lethargy, temperature increase from 38◦C, gastric reflux 5-10L...

  12. Age- and sex-associated effects on acute-phase proteins in Göttingen minipigs

    DEFF Research Database (Denmark)

    Christoffersen, Berit Ø.; Jensen, Søren Juel; Ludvigsen, Trine P.;

    2015-01-01

    Göttingen minipigs are a useful model for diseases having an inflammatory component, and the associated use of acute-phase proteins (APP) as biomarkers of inflammation warrants establishment of their reference ranges. The objective of this study was to establish reference values for selected APP......, haptoglobin and PAGP and a decrease in albumin. An acute LPS stimulus increased CRP, PMAP, haptoglobin, and SAA; PAGP was unchanged and albumin decreased. Obese pigs with and without diabetes showed increases in CRP and PAGP, albumin decreased, and haptoglobin and SAA were unchanged. PMAP was increased only...... and interpreting studies including aspects of inflammation. In addition, an APP response was verified after both acute and chronic stimuli....

  13. Age- and Sex-Associated Effects on Acute-Phase Proteins in Göttingen Minipigs

    DEFF Research Database (Denmark)

    Christoffersen, Berit Ø; Jensen, Søren J.; Ludvigsen, Trine P;

    2015-01-01

    Göttingen minipigs are a useful model for diseases having an inflammatory component, and the associated use of acute-phase proteins (APP) as biomarkers of inflammation warrants establishment of their reference ranges. The objective of this study was to establish reference values for selected APP......, haptoglobin and PAGP and a decrease in albumin. An acute LPS stimulus increased CRP, PMAP, haptoglobin, and SAA; PAGP was unchanged and albumin decreased. Obese pigs with and without diabetes showed increases in CRP and PAGP, albumin decreased, and haptoglobin and SAA were unchanged. PMAP was increased only...... and interpreting studies including aspects of inflammation. In addition, an APP response was verified after both acute and chronic stimuli....

  14. Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

    Directory of Open Access Journals (Sweden)

    Petra Procházková

    Full Text Available Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs of the 5'- or 3'-untranslated regions (UTR of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP. The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.

  15. CTL Responses to Regulatory Proteins Tat and Rev in HIV-1 B'/C Virus-Infected Individuals

    Institute of Scientific and Technical Information of China (English)

    MING-MING JIA; KUN-XUE HONG; JIAN-PING CHEN; HONG-WEI LIU; SHA LIU; XIAO-QING ZHANG; HONG-JING ZHAO; YI-MING SHAO

    2008-01-01

    To characterize HIV-1 specific CTL responses to regulatory proteins Tat and Rev in HIV-B'/C vires-infected ART-naive individuals. Methods HIV-1-specific CTL responses were analyzed by IFN-γ ELISPOT assay using overlapping peptides spanning the consensus sequences of HIV-1 clade C Tat and Rev proteins. Statistical analysis and graphical presentation were performed using SIGMAPLOT 10.0 and SIGMASTAT 3.5. For samples with a positive response, the magnitude of CTL responses was compared between HIV-1 C proteins by Wilcoxon rank sum test, and the significance threshold was P<0.05. Results Tat and Rev were frequently recognized, with 23% and 52% of the tested individuals having detectable responses to these proteins, respectively. Several immunodominant regions were detected in Rev. No significant correlation was observed between the magnitude and breadth of CTL responses to regulatory proteins and the control of virus replication in this study. Conclusion Tat and Rev can serve as targets for HIV-1-specific CTL, and several immunodominant regions are detectable in Rev. Further characterization of epitopes and their role in virus control may shed light on pathogenesis of HIV-1 natural infection and also be useful for the design and testing of candidate vaccines.

  16. Acute phencyclidine treatment induces extensive and distinct protein phosphorylation in rat frontal cortex

    DEFF Research Database (Denmark)

    Palmowski, Pawel; Rogowska-Wrzesinska, Adelina; Williamson, James

    2014-01-01

    assessment of the regulated proteins biological function revealed that PCP perturbs key processes in the frontal cortex including calcium homeostasis, organization of cytoskeleton, endo/exocytosis, and energy metabolism. This study on acute PCP treatment provides the largest proteomics and phosphoproteomics......Phencyclidine (PCP), a noncompetitive N-methyl-d-aspartate receptor antagonist, induces psychotomimetic effects in humans and animals. Administration of PCP to rodents is used as a preclinical model for schizophrenia; however, the molecular mechanisms underlying the symptoms remain largely unknown....... Acute PCP treatment rapidly induces behavioral and cognitive deficits; therefore, post-translational regulation of protein activity is expected to play a role at early time points. We performed mass-spectrometry- driven quantitative analysis of rat frontal cortex 15, 30, or 240 min after...

  17. Serum acute phase proteins in cows with SARA (Subacute Ruminal Acidosis suspect

    Directory of Open Access Journals (Sweden)

    C. Cannizzo

    2012-02-01

    Full Text Available The aim of this study was to evaluate the variations of Acute Phase Proteins (APPs and other blood constituents during the onset of the sub-acute ruminal acidosis (SARA pathological status. A total of 108 cows from 12 dairy herds were randomly selected and divided into three Groups of 36 animals each. All animals were subjected to a rumenocentesis. Group A was composed by subjects with a rumen pH>5.8, Group B was composed by subjects with a rumen pH ≤5.5≤5.8 and Group C was composed by subjects with a rumen pH<5.5. Blood samples were collected by jugular venipuncture and Haptoglobin (Hp, Serum Amyloid A (SAA, Total Proteins, Albumin and White Blood Cells (WBC were determined. One-way ANOVA showed a statistical significance on Rumen pH, Hp, SAA. SARA seems not stimulate the APPs production from liver.

  18. Recombinant scFv Antibodies against E Protein and N Protein of Severe Acute Respiratory Syndrome Virus

    Institute of Scientific and Technical Information of China (English)

    Hui LIU; Yan-Li DING; Wei HAN; Mei-Yun LIU; Rui-Yang TIAN; Sheng-Li YANG; Yi GONG

    2004-01-01

    Three single chain antibodies(scFv)against the proteins of severe acute respiratory syndrome coronavirus(SARS-CoV)were isolated by phage display from an scFv antibody library.Bio-panning was carried out against immobilized purified envelope(E)and nucleocapsid(N)proteins of SARS-CoV.Their binding activity and specificity to E or N protein of SARS-CoV were characterized by phage-ELISA.Two of them,B 10 and C20,could recognize non-overlapping epitopes of the E protein according to the two-site binding test result.Clone A 17 could recognize N protein.The sequence of the epitope or overlapping epitope of scFv antibody A17 was PTDSTDNNQNGGRNGARPKQRRPQ.The affinity(equilibrium dissociation constant,Kd)of SARS-CoV E protein was 5.7×10-8 M for B10 and 8.9×10-8 M for C20.The affinity of A17for N protein was 2.1 x 10-6 M.All three scFv antibodies were purified with affinity chromatography and determined by Western blot.

  19. Heat Shock Protein-27 Delays Acute Rejection After Cardiac Transplantation: An Experimental Model

    OpenAIRE

    2014-01-01

    Background Rejection is the major obstacle to survival after cardiac transplantation. We investigated whether overexpression of heat shock protein (Hsp)-27 in mouse hearts protects against acute rejection and the mechanisms of such protection. Methods Hearts from B10.A mice overexpressing human Hsp-27 (Hsp-27tg), or Hsp-27–negative hearts from littermate controls (LCs) were transplanted into allogeneic C57BL/6 mice. The immune response to B10.A hearts was investigated using quantitative polym...

  20. Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis.

    Directory of Open Access Journals (Sweden)

    Jose L Nieto-Torres

    2014-05-01

    Full Text Available Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV envelope (E gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS

  1. Protection effect of survivin protein overexpression on acute myocardial infarction in rats.

    Science.gov (United States)

    Yang, Meng; Li, Bo; Liu, Jingwei; Sun, Haiyan

    2015-01-01

    To investigate the protective effect of adenovirus mediated Survivin protein overexpression on acute myocardial infarction in rats. 45 acute myocardial infarction rat models were constructed by suture method and were randomly divided into sham group, model group and treatment group. The treatment group was injected with Survivin gene packed virus via ventricle. The model group was injected with equal titer of adenovirus packed empty vector. The sham group was not ligated. These rats were killed in 96 h after treatment. The levels of Survivin, Caspase-3, caspase-7 mRNA and protein in myocardial tissues were detected by real-time fluorescence quantitative PCR and Western blot. Myocardium tissue cell apoptosis were analyzed by TUNEL staining, the immunology of myocardial infarction tissue was analyzed by TTC staining. Compared with model group and sham group, the level of survivin protein in myocardium tissue of rats in treatment group was significantly increased (Pmyocardial tissue of rats in model group and treatment group were significantly increased, but the treatment group were significantly lower than those of model group (Pmyocardial infarction areas of rats in model group and treatment group were significantly higher than those of sham group, but the treatment group were significantly lower than those of model group (Pmyocardial tissue can significantly inhibit the expression of apoptosis promoting factor in myocardial tissue of acute myocardial infarction rats, reduce the apoptosis index of myocardial cells and the myocardial infarct size, which has great significance for protecting myocardial function.

  2. BAG1: The Guardian of Anti-Apoptotic Proteins in Acute Myeloid Leukemia

    OpenAIRE

    Sanja Aveic; Martina Pigazzi; Giuseppe Basso

    2011-01-01

    BCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, M...

  3. Acute differential effects of milk-derived dietary proteins on postprandial lipaemia in obese non-diabetic subjects

    DEFF Research Database (Denmark)

    Holmer-Jensen, J; Hartvigsen, M L; Mortensen, L S

    2012-01-01

    Postprandial lipaemia is an established risk factor for atherosclerosis. To investigate the acute effect of four milk-derived dietary proteins (alpha-lactalbumin, whey isolate, caseinoglycomacropeptide and whey hydrolysate) on postprandial lipaemia, we have conducted a randomized, acute, single-b......-blinded clinical intervention study with crossover design....

  4. Partitioning of genetic variation between regulatory and coding gene segments: the predominance of software variation in genes encoding introvert proteins.

    Science.gov (United States)

    Mitchison, A

    1997-01-01

    In considering genetic variation in eukaryotes, a fundamental distinction can be made between variation in regulatory (software) and coding (hardware) gene segments. For quantitative traits the bulk of variation, particularly that near the population mean, appears to reside in regulatory segments. The main exceptions to this rule concern proteins which handle extrinsic substances, here termed extrovert proteins. The immune system includes an unusually large proportion of this exceptional category, but even so its chief source of variation may well be polymorphism in regulatory gene segments. The main evidence for this view emerges from genome scanning for quantitative trait loci (QTL), which in the case of the immune system points to a major contribution of pro-inflammatory cytokine genes. Further support comes from sequencing of major histocompatibility complex (Mhc) class II promoters, where a high level of polymorphism has been detected. These Mhc promoters appear to act, in part at least, by gating the back-signal from T cells into antigen-presenting cells. Both these forms of polymorphism are likely to be sustained by the need for flexibility in the immune response. Future work on promoter polymorphism is likely to benefit from the input from genome informatics.

  5. Cloning, expression, purification and crystallization of a transcriptional regulatory protein (Rv3291c) from Mycobacterium tuberculosis H37Rv.

    Science.gov (United States)

    Shrivastava, Tripti; Kumar, Sandeep; Ramachandran, Ravishankar

    2004-10-01

    Rv3291c, the translational product of the Mycobacterium tuberculosis Rv3291c gene, is an 18 kDa protein. It is a putative transcriptional regulatory protein belonging to the leucine-responsive regulatory protein/asparagine synthase C (Lrp/AsnC) family, which are proteins that have been identified in archaea and bacteria. Rv3291c probably plays a significant role during the persistent/latent phase of M. tuberculosis, as supported by its up-regulation several-fold during this stage. Orthorhombic crystals of recombinant Rv3291c have been grown from trisodium citrate dihydrate-buffered solutions containing monoammonium dihydrogen phosphate. Diffraction data extending to 2.7 A have been collected from a single crystal with unit-cell parameters a = 99.6, b = 100.7, c = 100.6 A. Assuming an octamer in the asymmetric unit results in a Matthews coefficient (VM) of 1.75 A3 Da(-1), corresponding to a solvent content of about 30%.

  6. Pig-MAP, porcine acute phase proteins and standardisation of assays in Europe

    DEFF Research Database (Denmark)

    Alava, M.A.; Gonzalez-Ramon, N.; Heegaard, Peter M. H.;

    1997-01-01

    reactive protein (CRP) and haptoglobin (Hp) increase 5-7 times 48 h after the injection. Porcine Alb, alpha-lipoprotein, fetuin and transferrin were negative APP. Finally, the concentration of alpha(1)-acid glycoprotein and alpha(1)-protease inhibitor (alpha(1)-antitrypsin) did not change significantly......The pattern of plasma proteins changes greatly following infection, inflammation or tissue injury. The concentration of some proteins referred to as acute phase proteins (APPs) significantly increases within hours or days after the onset of these processes. In contrast, the concentration of other...... for the presence of infectious, inflammatory and pathological lesions in animals. The ability to monitor the APP concentration in serum of pigs will improve the quality and safety of the meat produced as well as provide important diagnostic information for animal health and welfare. The serum concentration of APP...

  7. Role of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in acute lung injury in rats

    DEFF Research Database (Denmark)

    Shanley, T P; Schmal, H; Friedl, H P

    1995-01-01

    The role of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in the pathogenesis of acute lung injury in rats after intrapulmonary deposition of IgG immune complexes or intratracheal administration of LPS has been assessed. Critical to these studies was the cloning and functional expression...... of rat MIP-1 alpha. The resulting product shared 92% and 90% homology with the known murine sequence at the cDNA level and protein level, respectively. Recombinant rat MIP-1 alpha exhibited dose-dependent chemotactic activity for both rat and human monocytes and neutrophils, which could be blocked...... by anti-murine MIP-1 alpha Ab. Rat MIP-1 alpha mRNA and protein expression were determined as a function of time in both injury models. A time-dependent increase in MIP-1 alpha mRNA in lung extracts was observed in both models. In the LPS model, MIP-1 alpha protein could also be detected...

  8. 14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

    DEFF Research Database (Denmark)

    Andersen, Sofie Dabros; Keijzers, Guido; Rampakakis, Emmanouil

    2012-01-01

    Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins...... experiments reveal weak affinity of the more selective isoform 14-3-3s but both 14-3-3 isoforms ¿ and s significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1...... are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding...

  9. Multiple drug resistance protein (MDR-1, multidrug resistance-related protein (MRP and lung resistance protein (LRP gene expression in childhood acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Elvis Terci Valera

    Full Text Available CONTEXT: Despite the advances in the cure rate for acute lymphoblastic leukemia, approximately 25% of affected children suffer relapses. Expression of genes for the multiple drug resistance protein (MDR-1, multidrug resistance-related protein (MRP, and lung resistance protein (LRP may confer the phenotype of resistance to the treatment of neoplasias. OBJECTIVE: To analyze the expression of the MDR-1, MRP and LRP genes in children with a diagnosis of acute lymphoblastic leukemia via the semiquantitative reverse transcription polymerase chain reaction (RT-PCR, and to determine the correlation between expression and event-free survival and clinical and laboratory variables. DESIGN: A retrospective clinical study. SETTING: Laboratory of Pediatric Oncology, Department of Pediatrics, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil. METHODS: Bone marrow aspirates from 30 children with a diagnosis of acute lymphoblastic leukemia were assessed for the expression of messenger RNA for the MDR-1, MRP and LRP genes by semi-quantitative RT-PCR. RESULTS: In the three groups studied, only the increased expression of LRP was related to worsened event-free survival (p = 0.005. The presence of the common acute lymphoblastic leukemia antigen (CALLA was correlated with increased LRP expression (p = 0.009 and increased risk of relapse or death (p = 0.05. The relative risk of relapse or death was six times higher among children with high LRP expression upon diagnosis (p = 0.05, as confirmed by multivariate analysis of the three genes studied (p = 0.035. DISCUSSION: Cell resistance to drugs is a determinant of the response to chemotherapy and its detection via RT-PCR may be of clinical importance. CONCLUSIONS: Evaluation of the expression of genes for resistance to antineoplastic drugs in childhood acute lymphoblastic leukemia upon diagnosis, and particularly the expression of the LRP gene, may be of clinical relevance, and should be the

  10. Immunodepletion of high-abundant proteins from acute and chronic wound fluids to elucidate low-abundant regulators in wound healing

    Directory of Open Access Journals (Sweden)

    Chojnacki Caroline

    2010-12-01

    Full Text Available Abstract Background The process of wound healing consists of several well distinguishable and finely tuned phases. For most of these phases specific proteins have been characterized, although the underlying mechanisms of regulation are not yet fully understood. It is an open question as to whether deficits in wound healing can be traced back to chronic illnesses such as diabetes mellitus. Previous research efforts in this field focus largely on a restricted set of marker proteins due to the limitations detection by antibodies imposes. For mechanistic purposes the elucidation of differences in acute and chronic wounds can be addressed by a less restricted proteome study. Mass spectrometric (MS methods, e.g. multi dimensional protein identification technology (MudPIT, are well suitable for this complex theme of interest. The human wound fluid proteome is extremely complex, as is human plasma. Therefore, high-abundant proteins often mask the mass spectrometric detection of lower-abundant ones, which makes a depletion step of such predominant proteins inevitable. Findings In this study a commercially available immunodepletion kit was evaluated for the detection of low-abundant proteins from wound fluids. The dynamic range of the entire workflow was significantly increased to 5-6 orders of magnitude, which makes low-abundant regulatory proteins involved in wound healing accessible for MS detection. Conclusion The depletion of abundant proteins is absolutely necessary in order to analyze highly complex protein mixtures such as wound fluids using mass spectrometry. For this the used immunodepletion kit is a first but important step in order to represent the entire dynamic range of highly complex protein mixtures in the future.

  11. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions

    DEFF Research Database (Denmark)

    Luo, Yonglun; Friis, Jenny Blechingberg; Fernandes, Ana Miguel;

    2015-01-01

    and involved in the human neurological diseases amyotrophic lateral sclerosis and fronto-temporal lobar degeneration. Results To determine the gene regulatory functions of FUS and EWS at the level of chromatin, we have performed chromatin immunoprecipitation followed by next generation sequencing (Ch...

  12. Stress-induced Start Codon Fidelity Regulates Arsenite-inducible Regulatory Particle-associated Protein (AIRAP) Translation*

    Science.gov (United States)

    Zach, Lolita; Braunstein, Ilana; Stanhill, Ariel

    2014-01-01

    Initial steps in protein synthesis are highly regulated processes as they define the reading frame of the translation machinery. Eukaryotic translation initiation is a process facilitated by numerous factors (eIFs), aimed to form a “scanning” mechanism toward the initiation codon. Translation initiation of the main open reading frame (ORF) in an mRNA transcript has been reported to be regulated by upstream open reading frames (uORFs) in a manner of re-initiation. This mode of regulation is governed by the phosphorylation status of eIF2α and controlled by cellular stresses. Another mode of translational initiation regulation is leaky scanning, and this regulatory process has not been extensively studied. We have identified arsenite-inducible regulatory particle-associated protein (AIRAP) transcript to be translationally induced during arsenite stress conditions. AIRAP transcript contains a single uORF in a poor-kozak context. AIRAP translation induction is governed by means of leaky scanning and not re-initiation. This induction of AIRAP is solely dependent on eIF1 and the uORF kozak context. We show that eIF1 is phosphorylated under specific conditions that induce protein misfolding and have biochemically characterized this site of phosphorylation. Our data indicate that leaky scanning like re-initiation is responsive to stress conditions and that leaky scanning can induce ORF translation by bypassing poor kozak context of a single uORF transcript. PMID:24898249

  13. Drug-drug interactions related to altered absorption and plasma protein binding: theoretical and regulatory considerations, and an industry perspective.

    Science.gov (United States)

    Hochman, Jerome; Tang, Cuyue; Prueksaritanont, Thomayant

    2015-03-01

    Drug-drug interactions (DDIs) related to altered drug absorption and plasma protein binding have received much less attention from regulatory agencies relative to DDIs mediated via drug metabolizing enzymes and transporters. In this review, a number of theoretical bases and regulatory framework are presented for these DDI aspects. Also presented is an industry perspective on how to approach these issues in support of drug development. Overall, with the exception of highly permeable and highly soluble (BCS 1) drugs, DDIs related to drug-induced changes in gastrointestinal (GI) physiology can be substantial, thus warranting more attentions. For a better understanding of absorption-associated DDI potential in a clinical setting, mechanistic studies should be conducted based on holistic integration of the pharmaceutical profiles (e.g., pH-dependent solubility) and pharmacological properties (e.g., GI physiology and therapeutic margin) of drug candidates. Although majority of DDI events related to altered plasma protein binding are not expected to be of clinical significance, exceptions exist for a subset of compounds with certain pharmacokinetic and pharmacological properties. Knowledge of the identity of binding proteins and the binding extent in various clinical setting (including disease states) can be valuable in aiding clinical DDI data interpretations, and ensuring safe and effective use of new drugs.

  14. Identification of Arsenic Direct-Binding Proteins in Acute Promyelocytic Leukaemia Cells

    Directory of Open Access Journals (Sweden)

    Tao Zhang

    2015-11-01

    Full Text Available The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS. More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1, heat shock 70 kDa protein 9 (HSPA9 and pyruvate kinase M2 (PKM2, were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL.

  15. Interaction of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Electrostatic sites on the type Ialpha regulatory subunit.

    Science.gov (United States)

    Gibson, R M; Ji-Buechler, Y; Taylor, S S

    1997-06-27

    Since a basic surface on the catalytic (C) subunit of cAMP-dependent protein kinase is important for binding to the regulatory (R) subunit, acidic residues in R were sought that might contribute to R-C interaction. Using differential labeling by a water-soluble carbodiimide (Buechler, T. A., and Taylor, S. S. (1990) Biochemistry 29, 1937-1943), seven specific carboxylates in RIalpha were identified that were protected from chemical modification in the holoenzyme; each was then replaced with Ala. Of these, rRI(E15A/E106A/D107A)), rRI(E105A), rRI(D140A), rRI(E143A), and rRI(D258A) all were defective in holoenzyme formation and define negative electrostatic surfaces on RIalpha. An additional conserved carboxylate, Glu101 in RIalpha and the equivalent, Glu99 in RIIalpha were mutated to Ala. Replacement of Glu101 had no effect while rRII(E99A) was very defective. RIalpha and RIIalpha thus differ in the molecular details of how they recognize C. Unlike wild-type RI, two additional mutants, rRI(D170A) and rRI(K242A), inhibited C-subunit stoichiometrically in the presence of cAMP and show increases in both on- and off-rates. Asp170, which contributes directly to the hydrogen bonding network in cAMP-binding site A, thus contributes also to holoenzyme stability.

  16. Sterol Regulatory Element Binding Protein 1a Regulates Hepatic Fatty Acid Partitioning by Activating Acetyl Coenzyme A Carboxylase 2

    OpenAIRE

    Im, Seung-Soon; Hammond, Linda E.; Yousef, Leyla; Nugas-Selby, Cherryl; Shin, Dong-Ju; Seo, Young-Kyo; Fong, Loren G.; Young, Stephen G.; Osborne, Timothy F.

    2009-01-01

    We generated a line of mice in which sterol regulatory element binding protein 1a (SREBP-1a) was specifically inactivated by insertional mutagenesis. Homozygous mutant mice were completely viable despite expressing SREBP-1a mRNA below 5% of normal, and there were minimal effects on expression of either SREBP-1c or -2. Microarray expression studies in liver, where SREBP-1a mRNA is 1/10 the level of the highly similar SREBP-1c, demonstrated that only a few genes were affected. The only downregu...

  17. Total leucocyte count, C-reactive protein and neutrophil count: Diagnostic Aid in acute appendicitis

    Directory of Open Access Journals (Sweden)

    Shafi Sheikh

    2009-01-01

    Full Text Available Background/Aim: Acute appendicitis is one of the most common acute intraabdominal affections seen in surgical departments, which can be treated easily if an accurate diagnosis is made in time. Otherwise, delay in diagnosis and treatment can lead to diffuse peritonitis. Materials and Methods: A study was conducted on 110 patients who were operated for acute appendicitis to determine the role and predictive value of the total leucocyte count (TLC, C-reactive protein (CRP and percentage of neutrophil count in the diagnosis of acute appendicitis. Preoperative TLC, CRP and percentage of neutrophil count were determined and were compared with the results of the histopathology of the removed appendix. Results: Of all the patients studied, 92 had histopathologically positive appendicitis. The TLC was found to be significantly high in 90 patients who proved to have acute appendicitis, whereas CRP was high in only 88 patients and neutrophil percentage was raised in 91; four had a normal CRP level. Thus, TLC had a sensitivity, specificity and positive predictive value of 97.82%, 55.55% and 91.8%, respectively. CRP had a sensitivity, specificity and positive predictive value of 95.6%, 77.77% and 95.6% respectively. Percentage of neutrophil count had a sensitivity, specificity and positive predictive value of 98.9%, 38.88% and 89.21%, respectively. When used in combination, there was a marked improvement in the specificity and the positive predictive value to 88.04% and 98.7%, respectively. Conclusion: The inflammatory markers, i.e., TLC, CRP and neutrophil count can be helpful in the diagnosis when measured together as this increases their specificity and positive predictive value.

  18. Role of macrophage chemoattractant protein-1 in acute inflammation after lung contusion.

    Science.gov (United States)

    Suresh, Madathilparambil V; Yu, Bi; Machado-Aranda, David; Bender, Matthew D; Ochoa-Frongia, Laura; Helinski, Jadwiga D; Davidson, Bruce A; Knight, Paul R; Hogaboam, Cory M; Moore, Bethany B; Raghavendran, Krishnan

    2012-06-01

    Lung contusion (LC), commonly observed in patients with thoracic trauma is a leading risk factor for development of acute lung injury/acute respiratory distress syndrome. Previously, we have shown that CC chemokine ligand (CCL)-2, a monotactic chemokine abundant in the lungs, is significantly elevated in LC. This study investigated the nature of protection afforded by CCL-2 in acute lung injury/acute respiratory distress syndrome during LC, using rats and CC chemokine receptor (CCR) 2 knockout (CCR2(-/-)) mice. Rats injected with a polyclonal antibody to CCL-2 showed higher levels of albumin and IL-6 in the bronchoalveolar lavage and myeloperoxidase in the lung tissue after LC. Closed-chest bilateral LC demonstrated CCL-2 localization in alveolar macrophages (AMs) and epithelial cells. Subsequent experiments performed using a murine model of LC showed that the extent of injury, assessed by pulmonary compliance and albumin levels in the bronchoalveolar lavage, was higher in the CCR2(-/-) mice when compared with the wild-type (WT) mice. We also found increased release of IL-1β, IL-6, macrophage inflammatory protein-1, and keratinocyte chemoattractant, lower recruitment of AMs, and higher neutrophil infiltration and phagocytic activity in CCR2(-/-) mice at 24 hours. However, impaired phagocytic activity was observed at 48 hours compared with the WT. Production of CCL-2 and macrophage chemoattractant protein-5 was increased in the absence of CCR2, thus suggesting a negative feedback mechanism of regulation. Isolated AMs in the CCR2(-/-) mice showed a predominant M1 phenotype compared with the predominant M2 phenotype in WT mice. Taken together, the above results show that CCL-2 is functionally important in the down-modulation of injury and inflammation in LC.

  19. Acute release of tissue-type plasminogen activator (t-PA) from the endothelium ; regulatory mechanisms and therapeutic target

    NARCIS (Netherlands)

    Schrauwen, Y.; Vries, R.E.M. de; Kooistra, T.; Emeis, J.J.

    1994-01-01

    The acute release of t-PA was studied in vitro in human endothelial cells from different origin. It proved possible to enhance the amounts of t-PA which can be released by increasing t-PA synthesis, both in vitro, and in vivo in rats. These data suggest the feasibility to induce and (or) enhance acu

  20. Regulatory pathways for ATP-binding cassette transport proteins in kidney proximal tubules

    NARCIS (Netherlands)

    Masereeuw, R.; Russel, F.G.M.

    2012-01-01

    The ATP-binding cassette transport proteins (ABC transporters) represent important determinants of drug excretion. Protective or excretory tissues where these transporters mediate substrate efflux include the kidney proximal tubule. Regulation of the transport proteins in this tissue requires elabor

  1. Acute guttate psoriasis patients have positive streptococcus hemolyticus throat cultures and elevated antistreptococcal M6 protein titers.

    Science.gov (United States)

    Zhao, Guang; Feng, Xiaoling; Na, Aihua; Yongqiang, Jiang; Cai, Qing; Kong, Jian; Ma, Huijun

    2005-02-01

    To further study the role of Streptococci hemolyticus infection and streptococcal M6 protein in the pathogenesis of acute guttate psoriasis, streptococcal cultures were taken from the throats of 68 patients with acute guttate psoriasis. PCR technique was applied to detect M6 protein encoding DNA from those cultured streptococci. Pure M6 protein was obtained by Sephacry/S-200HR and Mono-Q chromatography from proliferated Streptococcus hemolyticus. Antistreptococcal M6 protein titers were measured in the serum of patients with acute guttate psoriasis, plaque psoriasis and healthy controls by ELISA. A high incidence of Streptococcus hemolyticus culture was observed in the guttate psoriatic group compared with the plaque psoriasis and control groups. Fourteen strains of Streptococcus hemolyticus were cultured from the throats of 68 acute guttate psoriasis patients. Of these, 5 strains contain DNA encoding the M6 protein gene as confirmed by PCR technique. More than 85% purification of M6 protein was obtained from Streptococcus pyogenes. Applying our pure M6 protein with the ELISA methods, we found that the titer of antistreptococcal M6 protein was significantly higher in the serum of guttate psoriasis patients than in the control or plaque psoriasis groups (P psoriasis have a high incidence of Streptococcus hemolyticus in their throats and raised titers of antistreptococcal M6 protein in their sera.

  2. Identification of Novel Targets of the Human Cell Cycle Regulatory Protein Cdc34

    Science.gov (United States)

    1997-07-01

    human Cdc34 and its interacting proteins using Southern, Northern and Western blot analysis. 11 PROPRIETARY Conclusion Knowledge gained about...expression in yeast. Task 2: Month 2-3: Excision of the library (the prey) encoding candidate interacting proteins fused to the activation domain from...Cdc34 and its interacting proteins in carcinogenesis. Task 7: Month 18-28: Study of the structure of human CDC34 and its novel partner proteins in

  3. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Jamal, Shazia [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Levi, Edi [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Rishi, Arun K. [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); VA Medical Center, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Datta, Nabanita S., E-mail: ndatta@med.wayne.edu [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States)

    2013-07-12

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  4. Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions

    DEFF Research Database (Denmark)

    Di Roberto, Raphaël B; Chang, Belinda; Trusina, Ala;

    2016-01-01

    All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae...

  5. Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo

    Directory of Open Access Journals (Sweden)

    Victoria A. Meliopoulos

    2016-11-01

    Full Text Available The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1 capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2 capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3 from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction.

  6. BAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia.

    Science.gov (United States)

    Aveic, Sanja; Pigazzi, Martina; Basso, Giuseppe

    2011-01-01

    BCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance.

  7. BAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Sanja Aveic

    Full Text Available BCL2 associated Athano-Gene 1 (BAG1 is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance.

  8. Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans

    Directory of Open Access Journals (Sweden)

    Shakes Leighcraft A

    2012-09-01

    Full Text Available Abstract Background Non-coding DNA in and around the human Amyloid Precursor Protein (APP gene that is central to Alzheimer’s disease (AD shares little sequence similarity with that of appb in zebrafish. Identifying DNA domains regulating expression of the gene in such situations becomes a challenge. Taking advantage of the zebrafish system that allows rapid functional analyses of gene regulatory sequences, we previously showed that two discontinuous DNA domains in zebrafish appb are important for expression of the gene in neurons: an enhancer in intron 1 and sequences 28–31 kb upstream of the gene. Here we identify the putative transcription factor binding sites responsible for this distal cis-acting regulation, and use that information to identify a regulatory region of the human APP gene. Results Functional analyses of intron 1 enhancer mutations in enhancer-trap BACs expressed as transgenes in zebrafish identified putative binding sites of two known transcription factor proteins, E4BP4/ NFIL3 and Forkhead, to be required for expression of appb. A cluster of three E4BP4 sites at −31 kb is also shown to be essential for neuron-specific expression, suggesting that the dependence of expression on upstream sequences is mediated by these E4BP4 sites. E4BP4/ NFIL3 and XFD1 sites in the intron enhancer and E4BP4/ NFIL3 sites at −31 kb specifically and efficiently bind the corresponding zebrafish proteins in vitro. These sites are statistically over-represented in both the zebrafish appb and the human APP genes, although their locations are different. Remarkably, a cluster of four E4BP4 sites in intron 4 of human APP exists in actively transcribing chromatin in a human neuroblastoma cell-line, SHSY5Y, expressing APP as shown using chromatin immunoprecipitation (ChIP experiments. Thus although the two genes share little sequence conservation, they appear to share the same regulatory logic and are regulated by a similar set of transcription

  9. Comparative genomic analysis of two-component regulatory proteins in Pseudomonas syringae

    DEFF Research Database (Denmark)

    Lavin, J.L.; Kiil, Kristoffer; Resano, O.

    2007-01-01

    important differences in TCS proteins among the three P. syringae pathovars. Conclusion: In this article we present a thorough analysis of the identification and distribution of TCS proteins among the sequenced genomes of P. syringae. We have identified differences in TCS proteins among the three P...... requires a complex array of TCS proteins to cope with diverse plant hosts, host responses, and environmental conditions. Results: Based on the genomic data, pattern searches with Hidden Markov Model (HMM) profiles have been used to identify putative HKs and RRs. The genomes of Psy B728a, Pto DC3000 and Pph...... 1448A were found to contain a large number of genes encoding TCS proteins, and a core of complete TCS proteins were shared between these genomes: 30 putative TCS clusters, 11 orphan HKs, 33 orphan RRs, and 16 hybrid HKs. A close analysis of the distribution of genes encoding TCS proteins revealed...

  10. Loss of regulatory T cell function on anti-inflammation is correlated with increased risk of acute kidney injury development in patients with primary glomerulonephritis.

    Science.gov (United States)

    Dong, Qin; Cai, Chen; Gao, Feng; Xu, Ziqian; Fang, Yuan; Wang, Lijie; Cui, Jie

    2015-01-01

    Inflammation is believed to play a major role in the pathophysiology of acute kidney injury (AKI). The injury induces the generation of inflammatory mediators like cytokines and chemokines by tubular and endothelial cells which contribute to the recruiting of leukocytes into the kidneys. Early AKI risk evaluation is limited to demographic characteristics and past clinical histories, and no specific treatment is available. To better identify patients at risk of developing AKI, and devise more targeted treatment and prevention regimen, we tracked 158 primary glomerulonephritis patients for their occurrence of AKI, and analyzed the characteristics of their adaptive immune system. We found that in patients that later developed AKI, peripheral blood T cell composition is shifted toward IFN-g-producing Th1-like cells. While the composition of CD4(+)CD25(+) T cells were similar between patients that later developed AKI and patients without AKI development, in patients that later developed AKI, their CD4(+)CD25(+) T cells secreted less regulatory cytokine IL-10, and was unable to suppress proinflammatory cytokine production by CD4(+) T cells, while in patients without AKI development, CD4(+)CD25(+) T cells were able to suppress CD4(+) T cell-mediated IFN-g and IL-17 expression under stimulation, partially through IL-10 secretion. Collectively, we identified a defect in CD4(+)CD25(+) T cell regulatory function in patients at risk of developing AKI.

  11. The role of heparin-binding protein in acute respiratory distress syndrome

    Institute of Scientific and Technical Information of China (English)

    刘杨

    2014-01-01

    Objective To study the role of heparin-binding protein(HBP)in sepsis-associated acute respiratory distress syndrome(ARDS),and to evaluate the prognostic value of HBP in ARDS.Methods Sixty seven sepsis patients were enrolled in the prospective study.According to whether present ARDS,patients were divided into two groups:ARDS group and non-ARDS group.Blood samples were obtained within 2 hours after patients were diagnosed with sepsis.We measured the level of interleukin-6,interleukin-8 and HBP by ELISA,counted the

  12. [Determination of capillary plasma C-reactive protein during therapy for acute infectious lung diseases].

    Science.gov (United States)

    Makarenko, V V; Vavilikhina, N F; Kastrikina, T N; El'chaninova, S A

    2011-06-01

    Changes in the concentration of C-reactive protein (CRP), leukocytes, erythrocyte sedimentation rate, and differential blood count were comparatively estimated in the treatment of 66 infants (aged 1.12 +/- 0.95 years) with acute infectious lung diseases. There was a high correlation between capillary plasma and venous serum CRP concentrations. On the first day of effective antibiotic therapy, there was a significant decrease in CRP levels; the sensitivity and specificity were 96 and 94%, respectively. Thus, measurement of capillary blood CRP is an accessible and informative tool to monitor therapy for infectious lung diseases in infants.

  13. Optimal combinations of acute phase proteins for detecting infectious disease in pigs

    Directory of Open Access Journals (Sweden)

    Heegaard Peter MH

    2011-03-01

    Full Text Available Abstract The acute phase protein (APP response is an early systemic sign of disease, detected as substantial changes in APP serum concentrations and most disease states involving inflammatory reactions give rise to APP responses. To obtain a detailed picture of the general utility of porcine APPs to detect any disease with an inflammatory component seven porcine APPs were analysed in serum sampled at regular intervals in six different experimental challenge groups of pigs, including three bacterial (Actinobacillus pleuropneumoniae, Streptococcus suis, Mycoplasma hyosynoviae, one parasitic (Toxoplasma gondii and one viral (porcine respiratory and reproductive syndrome virus infection and one aseptic inflammation. Immunochemical analyses of seven APPs, four positive (C-reactive protein (CRP, haptoglobin (Hp, pig major acute phase protein (pigMAP and serum amyloid A (SAA and three negative (albumin, transthyretin, and apolipoprotein A1 (apoA1 were performed in the more than 400 serum samples constituting the serum panel. This was followed by advanced statistical treatment of the data using a multi-step procedure which included defining cut-off values and calculating detection probabilities for single APPs and for APP combinations. Combinations of APPs allowed the detection of disease more sensitively than any individual APP and the best three-protein combinations were CRP, apoA1, pigMAP and CRP, apoA1, Hp, respectively, closely followed by the two-protein combinations CRP, pigMAP and apoA1, pigMAP, respectively. For the practical use of such combinations, methodology is described for establishing individual APP threshold values, above which, for any APP in the combination, ongoing infection/inflammation is indicated.

  14. Acquired protein C deficiency in a child with acute myelogenous leukemia, splenic, renal, and intestinal infarction.

    Science.gov (United States)

    Farah, Roula A; Jalkh, Khalil S; Farhat, Hussein Z; Sayad, Paul E; Kadri, Adel M

    2011-03-01

    We report the case of a 6-year-old boy diagnosed with acute promyelocytic leukemia (AML-M3V) when he presented with pallor, abdominal pain, anorexia, and fatigue. Induction chemotherapy was started according to the AML-BFM 98 protocol along with Vesanoid (ATRA, All-trans retinoic acid). On the sixth day of induction, he developed splenic and gallbladder infarcts. Splenectomy and cholecystectomy were performed while chemotherapy induction continued as scheduled. Four days later, he developed ischemic areas in the kidneys and ischemic colitis in the sigmoid colon. Hypercoagulation studies showed severe deficiency of protein C. Tests showed protein C 16% (reference range 70-140%), protein S 87% (reference range 70-140%), antithrombin III 122% (reference range 80-120%), prothrombin time 13.6 s (reference = 11.3), INR (international normalized ratio) 1.21, partial thromboplastin time 33 s (reference = 33), fibrinogen 214 mg/dl, D-dimer 970 μg/ml, factor II 98%, and that antinuclear antibody, antiphospholipid antibodies, mutation for factor II gene (G20210A), and mutation for Arg506 Gln of factor V were all negative (factor V Leiden). There was no evidence of clinical disseminated intravascular coagulation (DIC). He was treated with low molecular weight heparin and did well. He continues to be in complete remission 7 years later with normal protein C levels. Acquired protein C deficiency can occur in a variety of settings and has been reported in acute myelocytic leukemia. However, clinically significant thrombosis in the absence of clinical DIC, such as our case, remains extremely rare.

  15. Sterol Regulatory Element Binding Protein (Srb1) Is Required for Hypoxic Adaptation and Virulence in the Dimorphic Fungus Histoplasma capsulatum

    Science.gov (United States)

    DuBois, Juwen C.; Smulian, A. George

    2016-01-01

    The Histoplasma capsulatum sterol regulatory element binding protein (SREBP), Srb1 is a member of the basic helix-loop-helix (bHLH), leucine zipper DNA binding protein family of transcription factors that possess a unique tyrosine (Y) residue instead of an arginine (R) residue in the bHLH region. We have determined that Srb1 message levels increase in a time dependent manner during growth under oxygen deprivation (hypoxia). To further understand the role of Srb1 during infection and hypoxia, we silenced the gene encoding Srb1 using RNA interference (RNAi); characterized the resulting phenotype, determined its response to hypoxia, and its ability to cause disease within an infected host. Silencing of Srb1 resulted in a strain of H. capsulatum that is incapable of surviving in vitro hypoxia. We found that without complete Srb1 expression, H. capsulatum is killed by murine macrophages and avirulent in mice given a lethal dose of yeasts. Additionally, silencing Srb1 inhibited the hypoxic upregulation of other known H. capsulatum hypoxia-responsive genes (HRG), and genes that encode ergosterol biosynthetic enzymes. Consistent with these regulatory functions, Srb1 silenced H. capsulatum cells were hypersensitive to the antifungal azole drug itraconazole. These data support the theory that the H. capsulatum SREBP is critical for hypoxic adaptation and is required for H. capsulatum virulence. PMID:27711233

  16. A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

    Science.gov (United States)

    Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi

    2014-05-01

    The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

  17. Homing in on Acute Graft vs. Host Disease: Tissue-Specific T Regulatory and Th17 Cells

    OpenAIRE

    Engelhardt, Brian G.; Crowe, James E.

    2010-01-01

    Acute graft vs. host disease (aGVHD) is a major limitation of hematopoietic stem cell transplantation (HSCT), and it causes significant morbidity and mortality for this patient population. This immune-mediated injury occurs unpredictably and is caused by donor-derived T cells reacting to recipient alloantigens. Although donor Th1 cells play a critical role in aGVHD generation, numerous arms of both the innate and the adaptive immune systems along with determinants of lymphocyte trafficking ar...

  18. Acute Phase Proteins in Response to Dictyocaulus viviparus Infection in Calves

    Directory of Open Access Journals (Sweden)

    Waller K Persson

    2004-06-01

    Full Text Available Three experiments were carried out to examine the acute phase response, as measured by the acute phase proteins (APP haptoglobin, serum amyloid A (SAA and fibrinogen, in calves infected with lungworm, Dictyocaulus vivparus. In addition, eosinophil counts were analysed. Three different dose models were used in 3 separate experiments: I 250 D. viviparus infective third stage larvae (L3 once daily for 2 consecutive days, II 100 D. viviparus L3 once daily for 5 consecutive days, and III 2000 L3 once. All 3 dose regimes induced elevated levels of haptoglobin, SAA and fibrinogen, although there was considerable variation both between and within experiments. A significant increase was observed in all 3 APP at one or several time points in experiment I and III, whereas in experiment II, the only significant elevation was observed for fibrinogen at one occasion. The eosinophil numbers were significantly elevated in all 3 experiments. The results show that lungworm infection can induce an acute phase response, which can be monitored by the selected APP. Elevated APP levels in combination with high numbers of eosinophils in an animal with respiratory disease may be used as an indicator of lung worm infection, and help the clinician to decide on treatment. However, high numbers of eosinophils and low levels of APP do not exclude a diagnosis of lungworm. Thus, lungworm infection may not be detected if measurements of APP are used to assess calf health in herds or individual animals.

  19. C-reactive protein as a risk factor for acute coronary syndrome

    Institute of Scientific and Technical Information of China (English)

    杨胜利; 何秉贤; 何作云; 张华; 何学兰; 张伟

    2002-01-01

    Objective We assessed thelevels of C-reactive protein ( CRP ) in patients with acute coronary syndrome (ACS) [including unstable angina pectoris (UAP), acute myocardial infarction (AMI) and sudden cardiac death (SCD) ] compared with non-ACS [including stable angina pectoris (SAP), old myocardial infarction (OMI) and healthy volunteers] and sought to test whether CRP are associated with clinical acute coronary syndrome. Methods Ultrasensitive immunoassay (rate nephelometry with the Beckman Array multitest immunoassay system) wasused to measure CRP levels in 91 patients with ACS(20 UAP, 71 AMI including 2 SCD) and non-ACS (34SAP, 25 patients with healing phase of AMI, 41 OMI and 94 control healthy subjects) Results CRP levels were higher in ACS group (18.50 + 23.98 mg/L [ SE 2.51, n = 91 ] ) compared with non - ACS group (3.89+7.14mg/L[SE0.51, n=194]) (P <0.01).Using Logistic Regression, CRP was a potent determinant of ACS ( OR = 1.65) Conclusion These results suggest that CRP has a strong association with ACS, and CRP is a risk factor of ACS.

  20. Functional Characterization of a Novel Pro-Apoptotic Transcriptional Regulatory Protein in Ovarian Cancer

    Science.gov (United States)

    2005-01-01

    initiated studies to elucidate PAPX interacting proteins using an antibody array as described below. Although in the initial proposal, we had proposed to...identify PAPX interacting proteins with other techniques such as yeast two hybrid and GST pulldown assays, we also tried the antibody array to achieve...the same goal and were successful in identifying PAPX interacting proteins . To address Aim #3, we utilized antibody array containing antibodies against

  1. Prediction value of the ratio of serum high-sensitivity C-reactive protein and prealbumin on acute myocardial infarction complicated with acute heart failure

    Institute of Scientific and Technical Information of China (English)

    Su-Yun Zu; Shuang Wang; Feng-Lan Yang; Bao-Gui Chen; Ming-Zhe Ma

    2015-01-01

    Objective:To through counting serum high-sensitivity C-reactive protein (hs-CRP), prealbumin (PAB) and the ratio of the two to analyze its predictive value on acute myocardial infarction complicated with postoperative acute heart failure.Methods: 100 cases of acute myocardial infarction patients treated in our hospital from January 2013 to January 2015 were selected as research objects. Serum PAB and hs-CRP values on the next day of admission as well as after PCI surgery were recorded respectively. Then PAB data change before and after surgery, PAB and hs-CRP change with or without postoperative cardiovascular events (heart failure) as well as the value of the ratio of hs-CRP and PAB on prediction of acute myocardial infarction complicated with acute heart failure was compared.Results:Postoperative PAB in 100 cases of myocardial infarction patients increased from (0.19±0.05) to (0.24±0.06), and the differences had statistical significance; 40 cases had postoperative cardiovascular events (heart failure), incidence being 40%; PAB in patients without postoperative cardiovascular events (heart failure) increased significantly, hs-CRP decreased significantly, and the differences had statistical significance; Logistic regression univariate analysis showed that acute myocardial infarction complicated with heart failure was associated with diabetes, PAB, hs-CRP and In (hs-CRP/PAB), and multivariate analysis showed that it was associated with In (hs-CRP/PAB). Conclusion:hs-CRP in patients with acute myocardial infarction increases, PAB decreases, postoperative PAB increases relatively, hs-CRP and PAB data shows different degrees of change with or without postoperative cardiovascular (heart failure), and In (hs-CRP/PAB) is associated with acute myocardial infarction complicated with heart failure and can be used as its prediction index.

  2. The onset of the progression of acute phase response mechanisms induced by extreme impacts can be followed by the decrease in blood levels of positive acute phase proteins.

    Science.gov (United States)

    Larina, Olga; Bekker, Anna

    Studies performed at space flights and earth-based simulation models detected the plasma indices of acute phase reaction (APR), i.e. the increase of APR cytokine mediators and alterations in the production of blood acute phase proteins (APP) at the initial stages of adaptation to altered gravity conditions. Acute phase response is the principal constituent of the functional activity of innate immunity system. Changes in plasma APPs contents are considered to serve the restoration of homeostasis state. According to trends of their concentration shifts at the evolving of acute phase reaction APPs are denoted as positive, neutral, or negative. Plasma concentrations of positive acute phase proteins α1-acid glycoprotein (α1-AGP), α1-antitrypsin (α1-AT), and neutral α2-macroglobulin (α2-M) were measured in human study at 12-hour antiorthostatic position (AOP) with 15° head down tilt and hypoxia experiments at 14% oxygen in pressure chamber. Both of these impacts were shown to produce alterations in the APP levels indicative for acute phase response. Nevertheless, in AOP experiment noticeable decrease in α1-AGP concentration occurred by hour 12, and even more pronounced decline of α1-AGP and α1-AT were found on hypoxia hours 12 and 36. Acute phase proteins α1-AGP and α2-M possess the features of proteinase inhibitors. This function is implemented by the formation of complexes with the molecules of proteolytic enzymes which subsequently are removed from the blood flow. Transient decrease in plasma concentrations of protease inhibitors on early phases of APR development was reported to result from the growth of plasma protease activity due to cathepsin release from activated leukocytes, which had not yet been compensated by enhanced APP synthesis. Being a carrier protein for positively charged and neutral substances, α1-AGP shows pronounced elevation in its blood content during APR development. As assumed, it is required for the transportation of the increased

  3. Lactoferrin acute-phase protein and proteinase inhibitors in blood of patients with Q-wave myocardial infraction complicated by acute heart failure

    Directory of Open Access Journals (Sweden)

    K. P. Belokoneva

    2012-01-01

    Full Text Available 153 patients with Q-wave noncomplicated and complicated myocardial infraction have been examined. Blood serum was studied for acute-phase proteins (alfa-2-macroglobulin (MG, alfa-1-antitrypsin (ATr, and lactoferrin (LF in 97 patients ((57.9 ± 1.06 years old. Blood was sampled at the 1st, 7th, and 14th days after myocardial infraction. The decrease of MG in acute phase was observed in patients with cardiogenic shock. Increased concentrations of LF and unchanged MG level at the 1—7 days were observed at pulmonary edema.

  4. Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles

    Science.gov (United States)

    Petrényi, Katalin; Molero, Cristina; Kónya, Zoltán; Erdődi, Ferenc; Ariño, Joaquin; Dombrádi, Viktor

    2016-01-01

    Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a

  5. Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B

    Science.gov (United States)

    Kong, Lingbao; Fujimoto, Akira; Nakamura, Mariko; Aoyagi, Haruyo; Matsuda, Mami; Watashi, Koichi; Suzuki, Ryosuke; Arita, Minetaro; Yamagoe, Satoshi; Dohmae, Naoshi; Suzuki, Takehiro; Sakamaki, Yuriko; Ichinose, Shizuko; Suzuki, Tetsuro; Wakita, Takaji

    2016-01-01

    ABSTRACT It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors. IMPORTANCE The hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication

  6. C-reactive protein as a predictor of adverse outcome in patients with acute coronary syndrome

    Directory of Open Access Journals (Sweden)

    A S Sheikh

    2012-01-01

    Full Text Available Background and Objectives: The acute-phase reactant C-reactive protein (CRP has been shown to reflect systemic and vascular inflammation and to predict future cardiovascular events. The objective of this study was to evaluate the prognostic value of CRP in predicting cardiovascular outcome in patients presenting with acute coronary syndromes. Patients and Methods: This prospective, single-centered study was carried out by the Department of Pathology in collaboration with the Department of Cardiology, Bolan Medical College Complex Quetta, Balochistan, Pakistan from January 2009 to December 2009. We studied 963 consecutive patients presenting with chest pain to Accident and Emergency Department. Patients were divided into four groups. Group-1 comprised patients with unstable angina; group-2 included patients with acute ST elevation myocardial infarction (STEMI; group-3 comprised patients with Non-ST elevation myocardial infarction (Non-STEMI and group-4 was the control group. All four groups were followed-up for 90 days for occurrence of cardiovascular events. Results: The CRP was elevated (>3 mg/L among 27.6% patients in Group-1; 70.9% in group- 2; 77.9% in group-3 and 5.3% in the control group. Among cases with elevated CRP, 92.1% had a cardiac event compared to 34.3% among patients with CRP £3 mg/L (P < 0.0001. The mortality was significantly higher (P < 0.0001 in group-2 (8.9% and group-3 (11.9% as compared to group-1 (2.1%. There was no cardiac event or mortality in Group-4. Conclusions: Elevated CRP is a predictor of adverse outcome in patients with acute coronary syndromes and helps in identifying patients who may be at risk of cardiovascular complications.

  7. Chemotherapeutic treatment reduces circulating levels of surfactant protein-D in children with acute lymphoblastic leukemia

    DEFF Research Database (Denmark)

    Rathe, Mathias; Sorensen, Grith L; Wehner, Peder S;

    2016-01-01

    BACKGROUND: Surfactant protein D (SP-D) is a host defense molecule of the innate immune system that enhances pathogen clearance and modulates inflammatory responses. We hypothesized that circulating SP-D levels are associated with chemotherapy-induced mucositis and infectious morbidity in children...... with acute lymphoblastic leukemia (ALL). PROCEDURE: In a prospective study, 43 children receiving treatment for ALL were monitored for mucosal toxicity from diagnosis through the induction phase of treatment. Serial blood draws were taken to determine the levels of SP-D, interleukin-6 (IL-6), C......-reactive protein, and white blood cells. Data on fever, antibiotics, and bacteremia were collected. Baseline levels of circulating SP-D were compared with healthy controls. RESULTS: Baseline values of circulating SP-D were similar to levels in healthy controls (median: 829 ng/ml vs. 657 ng/ml, respectively, P > 0...

  8. Cytoplasmic dynein and its regulatory proteins in Golgi pathology in nervous system disorders

    NARCIS (Netherlands)

    Jaarsma, Dick; Hoogenraad, Casper C

    2015-01-01

    The Golgi apparatus is a dynamic organelle involved in processing and sorting of lipids and proteins. In neurons, the Golgi apparatus is important for the development of axons and dendrites and maintenance of their highly complex polarized morphology. The motor protein complex cytoplasmic dynein has

  9. Cytoplasmic dynein and its regulatory proteins in Golgi pathology in nervous system disorders

    NARCIS (Netherlands)

    D. Jaarsma (Dick); C.C. Hoogenraad (Casper)

    2015-01-01

    textabstractThe Golgi apparatus is a dynamic organelle involved in processing and sorting of lipids and proteins. In neurons, the Golgi apparatus is important for the development of axons and dendrites and maintenance of their highly complex polarized morphology. The motor protein complex cytoplasmi

  10. Effects of competition on acute phase proteins and lymphocyte subpopulations - oxidative stress markers in eventing horses.

    Science.gov (United States)

    Valle, E; Zanatta, R; Odetti, P; Traverso, N; Furfaro, A; Bergero, D; Badino, P; Girardi, C; Miniscalco, B; Bergagna, S; Tarantola, M; Intorre, L; Odore, R

    2015-10-01

    The aim of the study was to evaluate markers of the acute phase response (APR) in eventing horses by measuring acute phase proteins (APP) (haptoglobin, Hp, and serum amyloid A, SAA), lysozyme, protein adducts such as pentosidine-like adducts (PENT), malondialdehyde adducts (MDA), hydroxynonenal adducts (HNE) and total advanced glycation/glycoxidation end products (AGEs), complete blood count and lymphocyte subpopulations (CD4+, CD8+ and CD21+) both at rest and at the end of an eventing competition. Blood samples were collected from eight Warmblood horses (medium age 10 ± 3) during an official national 2-day event competition at rest (R) and 10 min after the arrival of the cross-country test on the second day. Exercise caused a significant increase in red blood cell number, haemoglobin, packed cell volume, neutrophils, white blood cell and lymphocyte number; however, these values remained within the normal range. The CD4+ and CD8+ cells significantly increased, whereas the CD21+ lymphocytes decreased; a significant increase in serum SAA, lysozyme and protein carbonyl derivates was also observed. Two-day event causes significant changes in APR markers such as lysozyme, protein carbonyl derivates (HNE, AGEs, PENT) and lymphocyte subpopulations. The data support the hypothesis that 2-day event may alter significantly APR markers. Limitations of the study were the relatively small sample size and sampling time conditioned by the official regulations of the event. Therefore, further studies are needed to investigate the time required for recovery to basal values in order to define the possible effects on the immune function of the athlete horse.

  11. Acute phase protein expression during elephant endotheliotropic herpesvirus-1 viremia in Asian elephants (Elephas maximus).

    Science.gov (United States)

    Stanton, Jeffrey J; Cray, Carolyn; Rodriguez, Marilyn; Arheart, Kristopher L; Ling, Paul D; Herron, Alan

    2013-09-01

    Infection of Asian elephants (Elephas maximus) with elephant endotheliotropic herpesvirus (EEHV) can be associated with rapid, lethal hemorrhagic disease and has been documented in elephant herds in human care and in the wild. Recent reports describe real-time quantitative polymerase chain reaction (qPCR) assays used to monitor clinically ill elephants and also to detect subclinical EEHV1 infection in apparently healthy Asian elephants. Acute phase proteins have been demonstrated to increase with a variety of infectious etiologies in domesticated mammals but have not yet been described in elephants. In addition, the immune response of Asian elephants to EEHV1 infection has not been described. In this study, whole blood and trunk wash samples representing repeated measures from eight elephants were examined for the presence of EEHV1 using a qPCR assay. Elephants were classified into groups, as follows: whole blood negative and positive and trunk wash negative and positive. Serum amyloid A (SAA) and haptoglobin (HP) levels were compared between these groups. A significant difference in SAA was observed with nearly a threefold higher mean value during periods of viremia (P=0.011). Higher values of SAA were associated with >10,000 virus genome copies/ml EEHV1 in whole blood. There were no significant differences in HP levels, although some individual animals did exhibit increased levels with infection. These data indicate that an inflammatory process is stimulated during EEHV1 viremia. Acute phase protein quantitation may aid in monitoring the health status of Asian elephants.

  12. Structural Dynamics and Conformational Equilibria of SERCA Regulatory Proteins in Membranes by Solid-State NMR Restrained Simulations

    Science.gov (United States)

    De Simone, Alfonso; Mote, Kaustubh R.; Veglia, Gianluigi

    2014-01-01

    Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR parameters. Unlike the unrestrained simulations, the resulting sarcolipin structures are in agreement with distances and angles for hydrogen bonds in ideal helices. In the case of phospholamban, the restrained ensemble sampled the conformational interconversion between T (helical) and R (unfolded) states for the cytoplasmic region that could not be observed using standard structural refinements with the same experimental data set. This study underscores the importance of implementing NMR data in molecular dynamics protocols to better describe the conformational landscapes of membrane proteins embedded in realistic lipid membranes. PMID:24940774

  13. Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality

    DEFF Research Database (Denmark)

    Buchou, Thierry; Vernet, Muriel; Blond, Olivier

    2003-01-01

    Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene in m....... Thus, our study demonstrates that in mammals, CK2beta is essential for viability at the cellular level, possibly because it acquired new functions during evolution.......Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene...

  14. The mitochondrial PPR protein LOVASTATIN INSENSITIVE 1 plays regulatory roles in cytosolic and plastidial isoprenoid biosynthesis through RNA editing.

    Science.gov (United States)

    Tang, Jianwei; Kobayashi, Keiko; Suzuki, Masashi; Matsumoto, Shogo; Muranaka, Toshiya

    2010-02-01

    Unlike animals, plants synthesize isoprenoids via two pathways, the cytosolic mevalonate (MVA) pathway and the plastidial 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway. Little information is known about the mechanisms that regulate these complex biosynthetic networks over multiple organelles. To understand such regulatory mechanisms of the biosynthesis of isoprenoids in plants, we previously characterized the Arabidopsis mutant, lovastatin insensitive 1 (loi1), which is resistant to lovastatin and clomazone, specific inhibitors of the MVA and MEP pathways, respectively. LOI1 encodes a pentatricopeptide repeat (PPR) protein localized in mitochondria that is thought to have RNA binding ability and function in post-transcriptional regulation of mitochondrial gene expression. LOI1 belongs to the DYW subclass of PPR proteins, which is hypothesized to be correlated with RNA editing. As a result of analysis of RNA editing of mitochondrial genes in loi1, a defect in RNA editing of three genes, nad4, ccb203 and cox3, was identified in loi1. These genes are related to the respiratory chain. Wild type (WT) treated with some respiration inhibitors mimicked the loi1 phenotype. Interestingly, HMG-CoA reductase activity of WT treated with lovastatin combined with antimycin A, an inhibitor of complex III in the respiratory chain, was higher than that of WT treated with only lovastatin, despite the lack of alteration of transcript or protein levels of HMGR. These results suggest that HMGR enzyme activity is regulated through the respiratory cytochrome pathway. Although various mechanisms exist for isoprenoid biosynthesis, our studies demonstrate the novel possibility that mitochondrial respiration plays potentially regulatory roles in isoprenoid biosynthesis.

  15. Changes in protein expression in testes of L2 strain Taiwan country chickens in response to acute heat stress.

    Science.gov (United States)

    Wang, Shih-Han; Cheng, Chuen-Yu; Chen, Chao-Jung; Chen, Hsin-Hsin; Tang, Pin-Chi; Chen, Chih-Feng; Lee, Yen-Pai; Huang, San-Yuan

    2014-07-01

    Heat stress causes a decrease of fertility in roosters. Yet, the way acute heat stress affects protein expression remains poorly understood. This study investigated differential protein expression in testes of the L2 strain of Taiwan country chickens following acute heat stress. Twelve 45-week-old roosters were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2 hours of recovery, and with 6 hours of recovery. Testis samples were collected for morphologic assay and protein analysis. Some of the differentially expressed proteins were validated by Western blot and immunohistochemistry. Abnormal and apoptotic spermatogenic cells were observed at 2 hours of recovery after acute heat stress, especially among the spermatocytes. Two-dimensional difference gel electrophoresis revealed that 119 protein spots were differentially expressed in chicken testes following heat stress, and peptide mass fingerprinting revealed that these spots contained 92 distinct proteins. In the heat-stressed samples, the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated, and glutathione S-transferase, transgelin, and DJ-1 were upregulated. Our results demonstrate that acute heat stress impairs the processes of translation, protein folding, and protein degradation, and thus results in apoptosis and interferes with spermatogenesis. On the other hand, the increased expression of antioxidant enzymes, including glutathione S-transferase and DJ-1, may attenuate heat-induced damage. These findings may have implications for breeding chickens that can tolerate more extreme conditions.

  16. Comparative analysis of acute and chronic corticosteroid pharmacogenomic effects in rat liver: Transcriptional dynamics and regulatory structures

    Directory of Open Access Journals (Sweden)

    DuBois Debra C

    2010-10-01

    Full Text Available Abstract Background Comprehensively understanding corticosteroid pharmacogenomic effects is an essential step towards an insight into the underlying molecular mechanisms for both beneficial and detrimental clinical effects. Nevertheless, even in a single tissue different methods of corticosteroid administration can induce different patterns of expression and regulatory control structures. Therefore, rich in vivo datasets of pharmacological time-series with two dosing regimens sampled from rat liver are examined for temporal patterns of changes in gene expression and their regulatory commonalities. Results The study addresses two issues, including (1 identifying significant transcriptional modules coupled with dynamic expression patterns and (2 predicting relevant common transcriptional controls to better understand the underlying mechanisms of corticosteroid adverse effects. Following the orientation of meta-analysis, an extended computational approach that explores the concept of agreement matrix from consensus clustering has been proposed with the aims of identifying gene clusters that share common expression patterns across multiple dosing regimens as well as handling challenges in the analysis of microarray data from heterogeneous sources, e.g. different platforms and time-grids in this study. Six significant transcriptional modules coupled with typical patterns of expression have been identified. Functional analysis reveals that virtually all enriched functions (gene ontologies, pathways in these modules are shown to be related to metabolic processes, implying the importance of these modules in adverse effects under the administration of corticosteroids. Relevant putative transcriptional regulators (e.g. RXRF, FKHD, SP1F are also predicted to provide another source of information towards better understanding the complexities of expression patterns and the underlying regulatory mechanisms of those modules. Conclusions We have proposed a

  17. Selection on Coding and Regulatory Variation Maintains Individuality in Major Urinary Protein Scent Marks in Wild Mice.

    Directory of Open Access Journals (Sweden)

    Michael J Sheehan

    2016-03-01

    Full Text Available Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones.

  18. Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids

    Directory of Open Access Journals (Sweden)

    Sperry Ann O

    2008-01-01

    . TLRR is homologous to a class of regulatory subunits for PP1, a central phosphatase in the reversible phosphorylation of proteins that is key to modulation of many intracellular processes. TLRR may serve to target this important signaling molecule near the nucleus of developing spermatids in order to control the cellular rearrangements of spermiogenesis.

  19. A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB

    Science.gov (United States)

    Steer, Beatrix; Adler, Barbara; Jonjic, Stipan; Stewart, James P.; Adler, Heiko

    2010-01-01

    Background Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages. Methodology/Principal Findings Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation. Conclusions/Significance In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein. PMID:20657771

  20. The effect of transport stress on turkey (Meleagris gallopavo) liver acute phase proteins gene expression.

    Science.gov (United States)

    Marques, Andreia Tomás; Lecchi, Cristina; Grilli, Guido; Giudice, Chiara; Nodari, Sara Rota; Vinco, Leonardo J; Ceciliani, Fabrizio

    2016-02-01

    The aim of this study was to investigate the effects of transport-related stress on the liver gene expression of four acute phase proteins (APP), namely α1-acid glycoprotein (AGP), C-Reactive Protein (CRP), Serum Amyloid A (SAA) and PIT54, in turkeys (Meleagris gallopavo). A group of seven BUT BIG 6 commercial hens was subjected to a two-hour long road transportation and the quantitative gene expression of APP in the liver was compared to that of a non transported control group. The expression of AGP and CRP mRNA was found to be increased in animals slaughtered after road transport. The presence of AGP protein was also confirmed by immunohistochemistry and Western blotting. The results of this study showed that road-transport may induce the mRNA expression of immune related proteins. The finding that AGP and CRP can be upregulated during transport could suggest their use as for the assessment of turkey welfare during transport.

  1. Cell death induction by the acute promyelocytic leukemia-specific PML/RARα fusion protein

    Science.gov (United States)

    Ferrucci, Pier Francesco; Grignani, Francesco; Pearson, Mark; Fagioli, Marta; Nicoletti, Ildo; Pelicci, Pier Giuseppe

    1997-01-01

    PML/RARα is the abnormal protein product generated by the acute promyelocytic leukemia-specific t(15;17). Expression of PML/RARα in hematopoietic precursor cell lines induces block of differentiation and promotes survival. We report here that PML/RARα has a potent growth inhibitory effect on all nonhematopoietic cell lines and on the majority of the hematopoietic cell lines tested. Inducible expression of PML/RARα in fibroblasts demonstrated that the basis for the growth suppression is induction of cell death. Deletion of relevant promyelocytic leukemia (PML) and retinoic acid receptor (RARα) domains within the fusion protein revealed that its growth inhibitory effect depends on the integrity of the PML aminoterminal region (RING, B1, B2, and coiled coil regions) and the RARα DNA binding region. Analysis of the nuclear localization of the same PML/RARα deletion mutants by immunofluorescence and cell fractionation revealed that the biological activity of the fusion protein correlates with its microspeckled localization and its association to the nuclear matrix. The PML aminoterminal region, but not the RARα zinc fingers, is required for the proper nuclear localization of PML/RARα. We propose that the matrix-associated microspeckles are the active sites of PML/RARα and that targeting of RARα sequences to this specific nuclear subdomain through PML sequences is crucial to the activity of the fusion protein on survival regulation. PMID:9380732

  2. The RFA regulatory sequence-binding protein in the promoter of prostate-specific antigen gene

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found indispensable for androgen receptor (AR)-mediated transactivation of PSA promoter. In transfection and CAT assays, some nucleotides substitution in RFA could significantly decrease the androgen inducibility for PSA promoter. The in vitro DNA binding assay demonstrated that RFA bound specifically with some non-receptor protein factors in prostate cell nucleus, but the mutant type of RFA lost this ability, so RFA might be a novel accessory cis-element. The RFA-binding proteins were isolated and purified by affinity chromatography using RFA probes. SDS-PAGE and preliminary protein identification showed these proteins possessed sequence high homology with multifunctional protein heterogeneous nuclear ribonucleoprotein A1, A2 (hnRNP A1, A2). RFA-binding proteins possibly cooperate with AR-mediated transactivation for PSA promoter as coactivator. The study results will facilitate further understanding the mechanism and tissue specificity of PSA promoter.

  3. pH-Regulatory Proteins as Potential Targets in Breast Cancer

    DEFF Research Database (Denmark)

    Andersen, Anne Poder

    tissues. The focus of the present PhD study is on understanding the mechanisms through which pH-regulatory transporters are regulated by the breast tumor microenvironment, and how these transporters in turn favor cancer progression. In Paper I, we summarized the recent knowledge on the dynamic...... exhibit distinct spatial organization during 3D growth of MCF-7 and MDA-MB-231 breast cancer cells. By pharmacological inhibition and stable shRNA-mediated knockdown, we addressed the specific contributions of the transporters to spheroid growth and show that the specific transporters contribute to breast...... cancer spheroid growth in a cell-type dependent manner, with MCT1 and NBCn1 playing particular important roles in MCF-7 cells and NHE1 in MDA-MB-231 cells. In Papers III-IV we employed mouse models to study the functional relevance and the relative roles of NHE1, NBCn1 and MCT4 in breast cancer...

  4. Interleukin-1 family cytokines and their regulatory proteins in normal pregnancy and pre-eclampsia.

    Science.gov (United States)

    Southcombe, J H; Redman, C W G; Sargent, I L; Granne, I

    2015-09-01

    Maternal systemic inflammation is a feature of pre-eclampsia, a condition in pregnancy characterized by hypertension and proteinuria. Pre-eclampsia is caused by the placenta; many placental factors contribute to the syndrome's progression, and proinflammatory cytokines have been identified previously as one such mediator. The interleukin (IL)-1 family of cytokines are key regulators of the inflammatory network, and two naturally occurring regulatory molecules for IL-1 family cytokines, IL-1RA and sST2, have been found previously to be elevated in maternal blood from women with pre-eclampsia. Here we investigate more recently identified IL-1 family cytokines and regulatory molecules, IL-1RAcP, IL-37, IL-18BP, IL-36α/β/γ/Ra and IL-38 in pre-eclampsia. Pregnant women have more circulating IL-18BP and IL-36Ra than non-pregnant women, and sIL-1RAcP is elevated from women with pre-eclampsia compared to normal pregnancies. The placenta expresses all the molecules, and IL-37 and IL-18BP are up-regulated significantly in pre-eclampsia placentas compared to those from normal pregnancies. Together, these changes contribute to the required inhibition of maternal systemic cytotoxic immunity in normal pregnancy; however, in pre-eclampsia the same profile is not seen. Interestingly, the increased circulating levels of sIL-1RAcP and increased placental IL-18BP and IL-37, the latter of which we show to be induced by hypoxic damage to the placenta, are all factors which are anti-inflammatory. While the placenta is often held responsible for the damage and clinical symptoms of pre-eclampsia by the research community, here we show that the pre-eclampsia placenta is also trying to prevent inflammatory damage to the mother.

  5. Gamma glutamyltransferase levels and its association with high sensitive C-reactive protein in patients with acute coronary syndromes

    Science.gov (United States)

    Emiroglu, Mehmet Yunus; Esen, Özlem Batukan; Bulut, Mustafa; Karapinar, Hekim; Kaya, Zekeriya; Akcakoyun, Mustafa; Kargin, Ramazan; Aung, Soe Moe; Alızade, Elnur; Pala, Selcuk; Esen, Ali Metin

    2010-01-01

    Background: Elevated Gamma-glutamyltransferase (GGT) level is independently correlated with conditions associatedwith increased atherosclerosis, such as obesity, elevated serum cholesterol, high blood pressure and myocardial infarction. It is also demonstrated that serum gamma-glutamyltransferase activity is an independent risk factor for myocardial infarction and cardiac death in patients with coronary artery disease. Although the relationship between gamma-glutamyltransferase and coronary artery disease has been reported, not many studies have shown the relationship between changes ofgamma-glutamyltransferase in acute coronary syndromes and a well established coronary risk factor high sensitive C-reactive protein. (hs-CRP). Aims: In this study, how gamma-glutamyltransferase levels changed in acute coronary syndromes and its relationship with high sensitive C-reactive protein if any were studied. Patients & Methods: This trial was carried out at Kosuyolu Cardiovascular Training and Research Hospital and Van Yuksek Ihtisas Hospital, Turkey. 219 patients (177 males and 42 females) presenting with acute coronary syndrome, and 51 control subjects between September 2007 and September 2008 were included in the study. Serum gamma-glutamyltransferase, high sensitive C-reactive protein, serum lipoprotein levels and troponin I were determined. Results: Serum gamma-glutamyltransferase and high sensitive C-reactive protein levels were higher in acute coronary syndrome patients compared to control. There was also correlation between gamma-glutamyltransferase and high sensitive C-reactive protein levels. Conclusion: Serum gamma-glutamyltransferase and high sensitive C-reactive protein levels were higher in acute coronary syndrome patients. In subgroup analyses, the higher difference with Non-ST elevation myocardial infarction and ST elevation myocardial infarction groups than unstable angina oectoris group proposes a relationship between gamma-glutamyltransferase and severity

  6. Gamma glutamyltransferase levels and its association with high sensitive C-reactive protein in patients with acute coronary syndromes

    Directory of Open Access Journals (Sweden)

    Mehmet Yunus Emiroglu

    2010-07-01

    Full Text Available Background: Elevated Gamma-glutamyltransferase (GGT level is independently correlated with conditions associatedwith increased atherosclerosis, such as obesity, elevated serum cholesterol, high blood pressure and myocardial infarction. It is also demonstrated that serum gamma-glutamyltransferase activity is an independent risk factor for myocardial infarction and cardiac death in patients with coronary artery disease. Although the relationship between gamma-glutamyltransferase and coronary artery disease has been reported, not many studies have shown the relationship between changes ofgamma-glutamyltransferase in acute coronary syndromes and a well established coronary risk factor high sensitive C-reactive protein. (hs-CRP. Aims: In this study, how gamma-glutamyltransferase levels changed in acute coronary syndromes and its relationship with high sensitive C-reactive protein if any were studied. Patients & Methods:This trial was carried out at Kosuyolu Cardiovascular Training and Research Hospital and Van Yuksek Ihtisas Hospital, Turkey. 219 patients (177 males and 42 females presenting with acute coronary syndrome, and 51 control subjects between September 2007 and September 2008 were included in the study. Serum gamma-glutamyltransferase, high sensitive C-reactive protein, serum lipoprotein levels and troponin I were determined. Results: Serum gamma-glutamyltransferase and high sensitive C-reactive protein levels were higher in acute coronary syndrome patients compared to control. There was also correlation between gamma-glutamyltransferase and high sensitive C-reactive protein levels. Conclusion: Serum gamma-glutamyltransferase and high sensitive C-reactive protein levels were higher in acute coronary syndrome patients. In subgroup analyses, the higher difference with Non-ST elevation myocardial infarction and ST elevation myocardial infarction groups than unstable angina oectoris group proposes a relationship between gamma

  7. Gamma glutamyltransferase levels and its association with high sensitive C-reactive protein in patients with acute coronary syndromes

    Directory of Open Access Journals (Sweden)

    Mehmet Yunus Emiroglu

    2010-01-01

    Full Text Available Background: Elevated Gamma-glutamyltransferase (GGT level is independently correlated with conditions associatedwith increased atherosclerosis, such as obesity, elevated serum cholesterol, high blood pressure and myocardial infarction. It is also demonstrated that serum gamma-glutamyltransferase activity is an independent risk factor for myocardial infarction and cardiac death in patients with coronary artery disease. Although the relationship between gamma-glutamyltransferase and coronary artery disease has been reported, not many studies have shown the relationship between changes ofgamma-glutamyltransferase in acute coronary syndromes and a well established coronary risk factor high sensitive C-reactive protein. (hs-CRP. Aims: In this study, how gamma-glutamyltransferase levels changed in acute coronary syndromes and its relationship with high sensitive C-reactive protein if any were studied. Patients & Methods: This trial was carried out at Kosuyolu Cardiovascular Training and Research Hospital and Van Yuksek Ihtisas Hospital, Turkey. 219 patients (177 males and 42 females presenting with acute coronary syndrome, and 51 control subjects between September 2007 and September 2008 were included in the study. Serum gamma-glutamyltransferase, high sensitive C-reactive protein, serum lipoprotein levels and troponin I were determined. Results: Serum gamma-glutamyltransferase and high sensitive C-reactive protein levels were higher in acute coronary syndrome patients compared to control. There was also correlation between gamma-glutamyltransferase and high sensitive C-reactive protein levels. Conclusion: Serum gamma-glutamyltransferase and high sensitive C-reactive protein levels were higher in acute coronary syndrome patients. In subgroup analyses, the higher difference with Non-ST elevation myocardial infarction and ST elevation myocardial infarction groups than unstable angina oectoris group proposes a relationship between gamma

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

    Science.gov (United States)

    Shi, Tujin; Niepel, Mario; McDermott, Jason E; Gao, Yuqian; Nicora, Carrie D; Chrisler, William B; Markillie, Lye M; Petyuk, Vladislav A; Smith, Richard D; Rodland, Karin D; Sorger, Peter K; Qian, Wei-Jun; Wiley, H Steven

    2016-07-12

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components-16 core proteins and 10 feedback regulators-of the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling.

  9. Proteomic approach for acute-phase proteins of hemolymph and muscles in Scylla serrata challenged by a pathogenic bactedum

    Institute of Scientific and Technical Information of China (English)

    LIU Wenjie; SU Jing; WANG Guizhong; WANG Sanying

    2006-01-01

    Acute-phase response is documented to be a significant mechanism of innate immunity in vertebrates and invertebrates.In this study,proteomic methodologies were applied for different protein expressions in hemolymph of Scylla serrata challenged by Vibrio parahaemolyticus after immunization,and in muscles of the crabs separately challenged by V.parahaemolyticus.V.anguillarum and Aeromonas hydrophila.Up-regulated cryptocyanin is documented in the hemolymph and up-regulated calexcitin,wingless(fragment)and tachykinin-related peptide in the muscle as acute-phase proteins.All the four altered proteins were responsible for bacterial stress,but cryptocyanin seemed to be a memory response protein against the challenge by a live bacterium after immunization of the live cells.These up-regulated proteins can be indicative of an understanding of immunity of a crab.

  10. CD55 is a key complement regulatory protein that counteracts complement-mediated inactivation of Newcastle Disease Virus.

    Science.gov (United States)

    Rangaswamy, Udaya S; Cotter, Christopher R; Cheng, Xing; Jin, Hong; Chen, Zhongying

    2016-08-01

    Newcastle disease virus (NDV) is being developed as an oncolytic virus for virotherapy. In this study we analysed the regulation of complement-mediated inactivation of a recombinant NDV in different host cells. NDV grown in human cells was less sensitive to complement-mediated virus inactivation than NDV grown in embryonated chicken eggs. Additionally, NDV produced from HeLa-S3 cells is more resistant to complement than NDV from 293F cells, which correlated with higher expression and incorporation of complement regulatory proteins (CD46, CD55 and CD59) into virions from HeLa-S3 cells. Further analysis of the recombinant NDVs individually expressing the three CD molecules showed that CD55 is the most potent in counteracting complement-mediated virus inactivation. The results provide important information on selecting NDV manufacture substrate to mitigate complement-mediated virus inactivation.

  11. The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

    DEFF Research Database (Denmark)

    Yde, C W; Olsen, B B; Meek, D

    2008-01-01

    Cell-cycle transition from the G(2) phase into mitosis is regulated by the cyclin-dependent protein kinase 1 (CDK1) in complex with cyclin B. CDK1 activity is controlled by both inhibitory phosphorylation, catalysed by the Myt1 and Wee1 kinases, and activating dephosphorylation, mediated by the CDC...... interference results in delayed cell-cycle progression at the onset of mitosis. Knockdown of CK2beta causes stabilization of Wee1 and increased phosphorylation of CDK1 at the inhibitory Tyr15. PLK1-Wee1 association is an essential event in the degradation of Wee1 in unperturbed cell cycle. We have found...... regulatory subunit, identifying it as a new component of signaling pathways that regulate cell-cycle progression at the entry of mitosis.Oncogene advance online publication, 12 May 2008; doi:10.1038/onc.2008.146....

  12. The iron regulatory capability of the major protein participants in prevalent neurodegenerative disorders

    Directory of Open Access Journals (Sweden)

    Bruce Xue Wen Wong

    2014-04-01

    Full Text Available As with most bioavailable transition metals, iron is essential for many metabolic processes required by the cell but when left unregulated is implicated as a potent source of reactive oxygen species. It is uncertain whether the brain’s evident vulnerability to reactive species-induced oxidative stress is caused by a reduced capability in cellular response or an increased metabolic activity. Either way, dys-regulated iron levels appear to be involved in oxidative stress provoked neurodegeneration. As in peripheral iron management, cells within the central nervous system tightly regulate iron homeostasis via responsive expression of select proteins required for iron flux, transport and storage. Recently proteins directly implicated in the most prevalent neurodegenerative diseases, such as amyloid-β precursor protein, tau, α-synuclein, prion protein and huntingtin, have been connected to neuronal iron homeostatic control. This suggests that disrupted expression, processing or location of these proteins may result in a failure of their cellular iron homeostatic roles and augment the common underlying susceptibility to neuronal oxidative damage that is triggered in neurodegenerative disease.

  13. Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

    Directory of Open Access Journals (Sweden)

    L.M.P. Passaglia

    1998-11-01

    Full Text Available NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

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

    Science.gov (United States)

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

    2011-07-01

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

  15. Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense.

    Science.gov (United States)

    Passaglia, L M; Van Soom, C; Schrank, A; Schrank, I S

    1998-11-01

    NifA protein activates transcription of nitrogen fixation operons by the alternative sigma 54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

  16. Properties of the phage-shock-protein (Psp) regulatory complex that govern signal transduction and induction of the Psp response in Escherichia coli

    OpenAIRE

    Jovanovic, Goran; Engl, Christoph; Mayhew, Antony J.; Burrows, Patricia C.; Buck, Martin

    2010-01-01

    The phage-shock-protein (Psp) response maintains the proton-motive force (pmf) under extracytoplasmic stress conditions that impair the inner membrane (IM) in bacterial cells. In Escherichia coli transcription of the pspABCDE and pspG genes requires activation of σ 54-RNA polymerase by the enhancer-binding protein PspF. A regulatory network comprising PspF–A–C–B–ArcB controls psp expression. One key regulatory point is the negative control of PspF imposed by its binding to PspA. It has been p...

  17. Stress-evoked tyrosine phosphorylation of signal regulatory protein α regulates behavioral immobility in the forced swim test.

    Science.gov (United States)

    Ohnishi, Hiroshi; Murata, Takaaki; Kusakari, Shinya; Hayashi, Yuriko; Takao, Keizo; Maruyama, Toshi; Ago, Yukio; Koda, Ken; Jin, Feng-Jie; Okawa, Katsuya; Oldenborg, Per-Arne; Okazawa, Hideki; Murata, Yoji; Furuya, Nobuhiko; Matsuda, Toshio; Miyakawa, Tsuyoshi; Matozaki, Takashi

    2010-08-01

    Severe stress induces changes in neuronal function that are implicated in stress-related disorders such as depression. The molecular mechanisms underlying the response of the brain to stress remain primarily unknown, however. Signal regulatory protein alpha (SIRPalpha) is an Ig-superfamily protein that undergoes tyrosine phosphorylation and binds the protein tyrosine phosphatase Shp2. Here we show that mice expressing a form of SIRPalpha that lacks most of the cytoplasmic region manifest prolonged immobility (depression-like behavior) in the forced swim (FS) test. FS stress induced marked tyrosine phosphorylation of SIRPalpha in the brain of wild-type mice through activation of Src family kinases. The SIRPalpha ligand CD47 was important for such SIRPalpha phosphorylation, and CD47-deficient mice also manifested prolonged immobility in the FS test. Moreover, FS stress-induced tyrosine phosphorylation of both the NR2B subunit of the NMDA subtype of glutamate receptor and the K+-channel subunit Kvbeta2 was regulated by SIRPalpha. Thus, tyrosine phosphorylation of SIRPalpha is important for regulation of depression-like behavior in the response of the brain to stress.

  18. Functional analysis of membrane-bound complement regulatory protein on T-cell immune response in ginbuna crucian carp.

    Science.gov (United States)

    Nur, Indriyani; Abdelkhalek, Nevien K; Motobe, Shiori; Nakamura, Ryota; Tsujikura, Masakazu; Somamoto, Tomonori; Nakao, Miki

    2016-02-01

    Complements have long been considered to be a pivotal component in innate immunity. Recent researches, however, highlight novel roles of complements in T-cell-mediated adaptive immunity. Membrane-bound complement regulatory protein CD46, a costimulatory protein for T cells, is a key molecule for T-cell immunomodulation. Teleost CD46-like molecule, termed Tecrem, has been newly identified in common carp and shown to function as a complement regulator. However, it remains unclear whether Tecrem is involved in T-cell immune response. We investigated Tecrem function related to T-cell responses in ginbuna crucian carp. Ginbuna Tecrem (gTecrem) proteins were detected by immunoprecipitation using anti-common carp Tecrem monoclonal antibody (mAb) and were ubiquitously expressed on blood cells including CD8α(+) and CD4(+) lymphocytes. gTecrem expression on leucocyte surface was enhanced after stimulation with the T-cell mitogen, phytohaemagglutinin (PHA). Coculture with the anti-Tecrem mAb significantly inhibited the proliferative activity of PHA-stimulated peripheral blood lymphocytes, suggesting that cross-linking of Tecrems on T-cells interferes with a signal transduction pathway for T-cell activation. These findings indicate that Tecrem may act as a T-cell moderator and imply that the complement system in teleost, as well as mammals, plays an important role for linking adaptive and innate immunity.

  19. Xanthohumol Improves Diet-induced Obesity and Fatty Liver by Suppressing Sterol Regulatory Element-binding Protein (SREBP) Activation.

    Science.gov (United States)

    Miyata, Shingo; Inoue, Jun; Shimizu, Makoto; Sato, Ryuichiro

    2015-08-14

    Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that stimulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here, we demonstrate that a prenylated flavonoid in hops, xanthohumol (XN), is a novel SREBP inactivator that reduces the de novo synthesis of fatty acid and cholesterol. XN independently suppressed the maturation of SREBPs of insulin-induced genes in a manner different from sterols. Our results suggest that XN impairs the endoplasmic reticulum-to-Golgi translocation of the SREBP cleavage-activating protein (SCAP)-SREBP complex by binding to Sec23/24 and blocking SCAP/SREBP incorporation into common coated protein II vesicles. Furthermore, in diet-induced obese mice, dietary XN suppressed SREBP-1 target gene expression in the liver accompanied by a reduction of the mature form of hepatic SREBP-1, and it inhibited the development of obesity and hepatic steatosis. Altogether, our data suggest that XN attenuates the function of SREBP-1 by repressing its maturation and that it has the potential of becoming a nutraceutical food or pharmacological agent for improving metabolic syndrome.

  20. Xanthohumol Improves Diet-induced Obesity and Fatty Liver by Suppressing Sterol Regulatory Element-binding Protein (SREBP) Activation*

    Science.gov (United States)

    Miyata, Shingo; Inoue, Jun; Shimizu, Makoto; Sato, Ryuichiro

    2015-01-01

    Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that stimulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here, we demonstrate that a prenylated flavonoid in hops, xanthohumol (XN), is a novel SREBP inactivator that reduces the de novo synthesis of fatty acid and cholesterol. XN independently suppressed the maturation of SREBPs of insulin-induced genes in a manner different from sterols. Our results suggest that XN impairs the endoplasmic reticulum-to-Golgi translocation of the SREBP cleavage-activating protein (SCAP)-SREBP complex by binding to Sec23/24 and blocking SCAP/SREBP incorporation into common coated protein II vesicles. Furthermore, in diet-induced obese mice, dietary XN suppressed SREBP-1 target gene expression in the liver accompanied by a reduction of the mature form of hepatic SREBP-1, and it inhibited the development of obesity and hepatic steatosis. Altogether, our data suggest that XN attenuates the function of SREBP-1 by repressing its maturation and that it has the potential of becoming a nutraceutical food or pharmacological agent for improving metabolic syndrome. PMID:26140926

  1. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    Energy Technology Data Exchange (ETDEWEB)

    Lee, A L [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1996-05-01

    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the {delta}-Al-{var_epsilon} activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a {beta}{alpha}{beta}-{beta}{alpha}{beta} pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel {beta}-sheet. In addition {sup 15}N T{sub 1}, T{sub 2}, and {sup 15}N/{sup 1}H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone {sup 1}H, {sup 13}C, and {sup 15}N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and {sup 15}N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

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

    Energy Technology Data Exchange (ETDEWEB)

    Shi, T.; Niepel, M.; McDermott, J. E.; Gao, Y.; Nicora, C. D.; Chrisler, W. B.; Markillie, L. M.; Petyuk, V. A.; Smith, R. D.; Rodland, K. D.; Sorger, P. K.; Qian, W. -J.; Wiley, H. S.

    2016-07-12

    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 abundance 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.

  3. Identification of Novel Targets of the Human Cell Cycle Regulatory Protein Cdc34

    Science.gov (United States)

    1999-07-01

    results in a protein unable to complement a cdc34 mutation stages of spermatids and spermatozoa . In contrast, the repres- (34, 64). Comparable...described forms -> [ , i zn in Materials and Methods. Cotransfection of full-length hICERI3y (fly) or IgG heaivy pCS2MT-ICERIIy 1-33 (mini-lIy) with

  4. Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

    LENUS (Irish Health Repository)

    Whan, Vicki

    2010-11-23

    Abstract Background About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q) tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5) and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was identified

  5. Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

    Directory of Open Access Journals (Sweden)

    Barendse William

    2010-11-01

    Full Text Available Abstract Background About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5 and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was

  6. Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature.

    Science.gov (United States)

    Kleinschnitz, Christoph; Kraft, Peter; Dreykluft, Angela; Hagedorn, Ina; Göbel, Kerstin; Schuhmann, Michael K; Langhauser, Friederike; Helluy, Xavier; Schwarz, Tobias; Bittner, Stefan; Mayer, Christian T; Brede, Marc; Varallyay, Csanad; Pham, Mirko; Bendszus, Martin; Jakob, Peter; Magnus, Tim; Meuth, Sven G; Iwakura, Yoichiro; Zernecke, Alma; Sparwasser, Tim; Nieswandt, Bernhard; Stoll, Guido; Wiendl, Heinz

    2013-01-24

    We have recently identified T cells as important mediators of ischemic brain damage, but the contribution of the different T-cell subsets is unclear. Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) are generally regarded as prototypic anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. In the present study, we examined the role of Tregs after experimental brain ischemia/reperfusion injury. Selective depletion of Tregs in the DEREG mouse model dramatically reduced infarct size and improved neurologic function 24 hours after stroke and this protective effect was preserved at later stages of infarct development. The specificity of this detrimental Treg effect was confirmed by adoptive transfer experiments in wild-type mice and in Rag1(-/-) mice lacking lymphocytes. Mechanistically, Tregs induced microvascular dysfunction in vivo by increased interaction with the ischemic brain endothelium via the LFA-1/ICAM-1 pathway and platelets and these findings were confirmed in vitro. Ablation of Tregs reduced microvascular thrombus formation and improved cerebral reperfusion on stroke, as revealed by ultra-high-field magnetic resonance imaging at 17.6 Tesla. In contrast, established immunoregulatory characteristics of Tregs had no functional relevance. We define herein a novel and unexpected role of Tregs in a primary nonimmunologic disease state.

  7. Hyperlipidemia intensifies cerulein-induced acute pancreatitis associated with activation of protein kinase C in rats

    Institute of Scientific and Technical Information of China (English)

    Ya-Jun Wang; Jia-Bang Sun; Fei Li; Shu-Wen Zhang

    2006-01-01

    AIM: To investigate the effects of hyperlipidemia on acute pancreatitis (AP) and the possible mechanisms.METHODS: Rat models of hyperlipidemia and AP were established by Triton WR1339 and cerulein respectively.Human albumin was used to treat AP complicated by hyperlipidemia. In each group, we compared the histological score, volume of ascites, ratio of pancreatic wet/dry weight, serum amylase (AMY) and pancreatic acinar cell apoptosis. The level of protein kinase C (PKC) membrane translocation in pancreatic tissue was detected by Western blot.RESULTS: In the hyperlipidemia model established by Triton WR1339, triglyceride (TG) increased remarkably and reached its peak 6 h after injection, and most rats developed mild acute pancreatitis. Histological score, volume of ascites, ratio of wet/dry weight and serum AMY in AP animals with hyperlipidemia were obviously higher than those in AP animals (P <0.05) and decreased after albumin therapy but not significantly (P > 0.05). Apoptotic cells detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) increased in AP animals with hyperlipidemia and did not change distinctly after albumin therapy. PKC membrane translocation level increased in AP animals with hyperlipidemia and decreased remarkably after albumin therapy (P < 0.05).CONCLUSION: Hyperlipidemia may induce AP or intensify pancreatic injury. Albumin therapy can not alleviate pancreatic lesion effectively. PKC activation may be one mechanism by which AP is intensified by hyperlipidemia.

  8. INCREASE IN ACTIVATED PROTEIN C MEDIATES ACUTE TRAUMATIC COAGULOPATHY IN MICE

    Science.gov (United States)

    Chesebro, Brian B.; Rahn, Pamela; Carles, Michel; Esmon, Charles T.; Xu, Jun; Brohi, Karim; Frith, Daniel; Pittet, Jean-François; Cohen, Mitchell J.

    2013-01-01

    In severely injured and hypoperfused trauma patients, endogenous acute coagulopathy (EAC) is associated with an increased morbidity and mortality. Recent human data correlate this coagulopathy with activation of the protein C pathway. To examine the mechanistic role of protein C in the development of EAC, we used a mouse model of trauma and hemorrhagic shock, characterized by the combination of tissue injury and severe metabolic acidosis. Mice were subjected to one of four treatment groups: 1) C, control; 2) T, trauma (laparotomy); 3) H, hemorrhage (MAP, 35 mmHg × 60 min); 4) TH, trauma + hemorrhage. After 60 min, blood was drawn for analysis. Compared with C mice, the TH mice had a significantly elevated activated partial thromboplastin time (23.3 vs. 34.5 s) and significantly increased levels of activated protein C (aPC; 2.30 vs. 13.58 ng/mL). In contrast, T and H mice did not develop an elevated activated partial thromboplastin time or increased aPC. Selective inhibition of the anticoagulant property of aPC prevented the coagulopathy seen in response to trauma/hemorrhage (23.5 vs. 38.6 s [inhibitory vs. control monoclonal antibody]) with no impact on survival during the shock period. However, complete blockade of both the anticoagulant and cytoprotective functions of aPC caused 100% mortality within 45 min of shock, with histopathology evidence of pulmonary thrombosis and perivascular hemorrhage. These results indicate that our unique mouse model of T/H shock mimics our previous observations in trauma patients and demonstrates that EAC is mediated by the activation of the protein C pathway. In addition, the cytoprotective effect of protein C activation seems to be necessary for survival of the initial shock injury. PMID:19333141

  9. Acute-phase protein behavior in dairy cattle herd naturally infected with Trypanosoma vivax.

    Science.gov (United States)

    Sampaio, Paulo Henrique; Fidelis Junior, Otavio Luiz; Marques, Luiz Carlos; Machado, Rosangela Zacarias; Barnabé, Patrícia de Athayde; André, Marcos Rogério; Balbuena, Tiago Santana; Cadioli, Fabiano Antonio

    2015-07-30

    Trypanosoma vivax is a hemoprotozoon that causes disease in cattle and is difficult to diagnose. The host-parasite relationship in cattle that are infected by T. vivax has only been poorly studied. In the present study, a total of 429 serum proteinograms were produced from naturally infected animals (NIF) and were compared with 50 samples from control animals (C). The total protein, IgA band, complement C3 β chain band, albumin band, antitrypsin band, IgG band, haptoglobin band, complement C3c α chain band and protein HP-20 band presented higher levels in the serum proteinograms of the NIF group. Inter-alpha-trypsin inhibitor heavy chain H4, α2-macroglobulin, complement C6, ceruloplasmin, transferrin band and apolipoprotein A1 band presented lower levels in this group. There was no significant difference (pNIF and C groups. Acute phase proteins may be useful for understanding the host-parasite relationship, since the antitrypsin band was only present in the NIF group. This can be used as an indicator for infection in cattle that are naturally infected by T. vivax.

  10. Changes of pregnancy-associated plasma protein-A in patients with acute coronary syndrome

    Institute of Scientific and Technical Information of China (English)

    LIU Jin-lai; ZHANG Hui; XIE Xu-jing; CHEN Lin; ZHAO Chang-lin

    2005-01-01

    @@ The term vulnerable patient has been proposed to define subjects susceptible to an acute coronary syndrome (ACS) or sudden cardiac death based on plaque characteristics, blood abnorma-lities, or myocardial vulnerability.1 It will be important in the future to identify both vulnerable patients and vulnerable plaques. Atherosclerotic arteries obtained at autopsy from patients who died suddenly of cardiac causes indicate that pregnancy-associated plasma protein-A (PAPP-A) was abundantly expressed in plaque cells and in the extracellular matrix of ruptured and eroded unstable plaques, but not in stable plaques.2 Here we examined circulating PAPP-A levels in patients with ACS in order to evaluate its potential use in identifying vulnerable patients.

  11. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Andrew Loyd [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1996-05-01

    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the δ-Al-ε activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a βαβ-βαβ pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel β-sheet. In addition 15N T1, T2, and 15N/1H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone 1H, 13C, and 15N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and 15N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

  12. The Significance and Correlation of SODD and Bcl-2 Protein Expression in Acute Leukemia of Children

    Institute of Scientific and Technical Information of China (English)

    Hongfang Tao; Qun Hu; Liuqing Zhang; Aiguo Liu; Shuangyou Liu; Ying Hu

    2006-01-01

    OBJECTIVE To explore the expression of SODD and bcl-2 proteins in bone marrow cells of children with acute leukemia (AL), and to examine the relationship of their expression with the classification, clinical features,therapeutic effect and prognosis for AL patients.METHODS Using the SABC immunohistochemical staining method, the expression of SODD and bcl-2 proteins in the bone marrow cells of 86 AL cases was determined. The patients were studied based on the following groups: 1) a first-visiting group; 2) a refractory-relapse group(some patients were sensitive to therapy but then suffered a recurrence);3) a complete-remission group (CR); 4) a high risk (HR) and 5) standard risk (SR) group; 6) a control group of patients with non-hematological diseases.RESULTS The positive rates of SODD and bcl-2 expression in the firstvisit, refractory-relapse and CR groups were significantly higher (P<0.05)compared to the control group. There was no significant difference in the expression of SODD or bcl-2 proteins between an acute lymphoblastic leukemia (ALL) group and acute nonlymphoblastic leukemia (ANLL)group (t=1.874, t=1.583, P>0.05). The positive rates of SODD and bcl-2expression in the patients who developed complete remission after chemotherapy were significantly lower (t=2.054, t=2.703, P<0.05) compared to the first-visit pediatric patients. The expression of the SODD protein in the refractory-relapse group was notably higher compared to the group treated initially (t=-1.081, P<0.05). A high expression of the bcl-2 protein was found in both the first-visit and refractory-relapse groups, with no significant difference found between the two groups (t=-1.196, P>0.05), whereas the percentage of bcl-2 positive cells in the refractory-relapse group (45%~87%) was significantly higher compared to the first-visit group (5%~62%). The positive expression of the SODD and bcl-2 proteins in the high-risk (HR) group were both significantly higher than the SR group (t=-3

  13. Control of Vibrio fischeri lux gene transcription by a cyclic AMP receptor protein-luxR protein regulatory circuit.

    OpenAIRE

    1988-01-01

    Expression of the Vibrio fischeri luminescence genes (lux genes) requires two transcriptional activators: the V. fischeri luxR gene product with autoinducer and the cyclic AMP (cAMP) receptor protein (CRP) with cAMP. It has been established that autoinducer and the luxR gene product are required for transcriptional activation of the luxICDABE operon, which contains a gene required for autoinducer synthesis and genes required for light emission. However, the role of cAMP-CRP in the induction o...

  14. Interactions between Barley a-Amylases, Substrates, Inhibitors and Regulatory Proteins

    DEFF Research Database (Denmark)

    Hachem, Maher Abou; Bozonnet, Sophie; Willemoës, Martin

    2006-01-01

    Barley a-amylase binds sugars at two sites on the enzyme surface in addition to the active site. Crystallography and site-directed mutagenesis highlight the importance of aromatic residues at these surface sites as demonstrated by Kd values determined for ß-cyclodextrin by surface plasmon resonance......, a fully hydrated calcium ion at the protein interface mediates contact between inhibitor residues and the enzyme catalytic groups in a manner that depends on calcium and which can be suppressed by site-directed mutagenesis of Glu168 in BASI. Finally certain inhibitors and enzymes are targets...... of the disulphide reductase thioredoxin h that attacks a specific disulphide bond in BASI and, remarkably, reduces two different disulphide bonds in the barley monomeric and dimeric amylase inhibitors that both belong to the CM-proteins and inhibit animal a-amylase....

  15. Regulatory effect of heat shock protein 70 in stress-induced rat intestinal epithelial barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Stevie Struiksma

    2009-06-01

    Full Text Available Background: Psychological stress is one of the factors associated with many human diseases; the mechanisms need to be further understood. Methods: Rats were subjected to chronic water avoid stress. Intestinal epithelial heat shock protein (HSP 70 was evaluated. The intestinal epithelial permeability was examined with Ussing chamber technique. Results: HSP70 was detected in normal intestinal epithelial cells. Psychological stress decreased HSP70 in the intestinal epithelial cells that correlated with the stress-induced intestinal epithelial hyperpermeability. Pretreatment with HSP70 abrogated stress-induced intestinal barrier dysfunction. Conclusions: Chronic stress inhibits HSP70 activity in rat intestinal epithelial layer that is associated with intestinal epithelial barrier dysfunction, which can be prevented by pretreatment with HSP70 protein.

  16. Expression of survivin, a novel apoptosis inhibitor and cell cycle regulatory protein, in human gliomas

    Institute of Scientific and Technical Information of China (English)

    焦保华; 姚志刚; 耿少梅; 左书浩

    2004-01-01

    @@ Recently, a novel anti-apoptosis gene, named survivin,was identified as a structurally unique member of the inhibitor of apoptosis protein (lAP) family. The gene is located on chromosome 17q25. Survivin is a 16.5 kDa protein that is expressed in vivo in common human cancers, but not in normal adjacent tissue,1 during the G2/M phase of the cell cycle. Survivin expression is turned off during fetal development and not found in nonneoplastic adult human tissue, and it is turned on in most common human cancers. We investigated the expression of survivin in 50 patients with human gliomas, and determined its association with cell apoptosis and cell proliferation, and its impact on tumor progression and prognosis.

  17. Functional and Developmental Analysis of CD4+CD25+ Regulatory T Cells under the Influence of Streptococcal M Protein in Rheumatic Heart Disease

    Directory of Open Access Journals (Sweden)

    Nidhal Abdul-Auhaimen

    2011-06-01

    Full Text Available The purpose of this study was to determine the role of streptococcal M protein in naturally-occurring CD4+CD25+ regulatory T cells (nTregs function and development in rheumatic heart disease in Iraqi patients. Streptococcus pyogenes was isolated for subsequent M protein extraction. Also, peripheral blood nTregs and CD4+ T cells were isolated by using Magnetic Cell Separation System. Tissue culture for isolated cells was performed in the presence and absence of M protein. Cell count was performed, and tumor necrosis factor alpha (TNF-α and interleukin-4 (IL-4 were determined in culture supernatant using ELISA system. There was a significant positive correlation (P0.05, association (r=0.353 between the mean number of nTregs and CD4+ T cells in the presence of M protein. The M protein stimulated CD4+ T cells to produce IL-4 in very little amount (<4 pg/ml in all samples. Compared to the production of IL4, TNF-α was produced in higher concentrations in the culture supernatants. The findings of the study indicate that streptococcal M protein has an important role in increasing the proliferation of CD4+CD25+regulatory T cells and CD4+ T cells. However, CD4+CD25+ regulatory T cells have lower suppressive activity against CD4+ T cells in the presence of M protein

  18. Functional and Developmental Analysis of CD4+CD25+ Regulatory T Cells under the Influence of Streptococcal M Protein in Rheumatic Heart Disease

    Science.gov (United States)

    Abdul-Auhaimena, Nidhal; Al-Kaabi, Zaman I. L

    2011-01-01

    The purpose of this study was to determine the role of streptococcal M protein in naturally-occurring CD4+CD25+ regulatory T cells (nTregs) function and development in rheumatic heart disease in Iraqi patients. Streptococcus pyogenes was isolated for subsequent M protein extraction. Also, peripheral blood nTregs and CD4+ T cells were isolated by using Magnetic Cell Separation System. Tissue culture for isolated cells was performed in the presence and absence of M protein. Cell count was performed, and tumor necrosis factor alpha (TNF-α) and interleukin-4 (IL-4) were determined in culture supernatant using ELISA system. There was a significant positive correlation (P0.05), association (r=0.353) between the mean number of nTregs and CD4+ T cells in the presence of M protein. The M protein stimulated CD4+ T cells to produce IL-4 in very little amount (<4 pg/ml) in all samples. Compared to the production of IL4, TNF-α was produced in higher concentrations in the culture supernatants. The findings of the study indicate that streptococcal M protein has an important role in increasing the proliferation of D4+CD25+regulatory T cells and CD4+ T cells. However, CD4+CD25+ regulatory T cells have lower suppressive activity against CD4+ T cells in the presence of M protein. PMID:23359747

  19. Lamins, laminopathies and disease mechanisms: Possible role for proteasomal degradation of key regulatory proteins

    Indian Academy of Sciences (India)

    Veena K Parnaik; Pankaj Chaturvedi; B H Muralikrishna

    2011-08-01

    Lamins are major structural proteins of the nucleus and are essential for nuclear integrity and organization of nuclear functions. Mutations in the human lamin genes lead to highly degenerative genetic diseases that affect a number of different tissues such as muscle, adipose or neuronal tissues, or cause premature ageing syndromes. New findings on the role of lamins in cellular signalling pathways, as well as in ubiquitin-mediated proteasomal degradation, have given important insights into possible mechanisms of pathogenesis.

  20. Functional Characterization of a Novel Pro-Apoptotic Transcription Regulatory Protein in Ovarian Cancer

    Science.gov (United States)

    2006-12-01

    Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES Original contains colored plates: ALL DTIC reproductions will be in...region of NADE that is shown to be involved in NGF -dependent regulation of NADE-induced apoptosis [4]. NADE is a nuclear pro-apoptotic protein...and NADE share high degree of homology except between residues 72-112 of NADE, a region essential for NGF -dependent regulation of NADE-induced

  1. Transcriptional analysis of the jamaicamide gene cluster from the marine cyanobacterium Lyngbya majuscula and identification of possible regulatory proteins

    Directory of Open Access Journals (Sweden)

    Dorrestein Pieter C

    2009-12-01

    Full Text Available Abstract Background The marine cyanobacterium Lyngbya majuscula is a prolific producer of bioactive secondary metabolites. Although biosynthetic gene clusters encoding several of these compounds have been identified, little is known about how these clusters of genes are transcribed or regulated, and techniques targeting genetic manipulation in Lyngbya strains have not yet been developed. We conducted transcriptional analyses of the jamaicamide gene cluster from a Jamaican strain of Lyngbya majuscula, and isolated proteins that could be involved in jamaicamide regulation. Results An unusually long untranslated leader region of approximately 840 bp is located between the jamaicamide transcription start site (TSS and gene cluster start codon. All of the intergenic regions between the pathway ORFs were transcribed into RNA in RT-PCR experiments; however, a promoter prediction program indicated the possible presence of promoters in multiple intergenic regions. Because the functionality of these promoters could not be verified in vivo, we used a reporter gene assay in E. coli to show that several of these intergenic regions, as well as the primary promoter preceding the TSS, are capable of driving β-galactosidase production. A protein pulldown assay was also used to isolate proteins that may regulate the jamaicamide pathway. Pulldown experiments using the intergenic region upstream of jamA as a DNA probe isolated two proteins that were identified by LC-MS/MS. By BLAST analysis, one of these had close sequence identity to a regulatory protein in another cyanobacterial species. Protein comparisons suggest a possible correlation between secondary metabolism regulation and light dependent complementary chromatic adaptation. Electromobility shift assays were used to evaluate binding of the recombinant proteins to the jamaicamide promoter region. Conclusion Insights into natural product regulation in cyanobacteria are of significant value to drug discovery

  2. Increase in cell viability by polyamines through stimulation of the synthesis of ppGpp regulatory protein and ω protein of RNA polymerase in Escherichia coli.

    Science.gov (United States)

    Terui, Yusuke; Akiyama, Mariko; Sakamoto, Akihiko; Tomitori, Hideyuki; Yamamoto, Kaneyoshi; Ishihama, Akira; Igarashi, Kazuei; Kashiwagi, Keiko

    2012-02-01

    It is known that polyamines increase cell growth through stimulation of the synthesis of several kinds of proteins encoded by the so-called "polyamine modulon". We recently reported that polyamines also increase cell viability at the stationary phase of cell growth through stimulation of the synthesis of ribosome modulation factor, a component of the polyamine modulon. Accordingly, we looked for other proteins involved in cell viability whose synthesis is stimulated by polyamines. It was found that the synthesis of ppGpp regulatory protein (SpoT) and ω protein of RNA polymerase (RpoZ) was stimulated by polyamines at the level of translation. Stimulation of the synthesis of SpoT and RpoZ by polyamines was due to an inefficient initiation codon UUG in spoT mRNA and an unusual location of a Shine-Dalgarno (SD) sequence in rpoZ mRNA. Accordingly, the spoT and rpoZ genes are components of the polyamine modulon involved in cell viability. Reduced cell viability caused by polyamine deficiency was prevented by modified spoT and rpoZ genes whose synthesis was not influenced by polyamines. Under these conditions, the level of ppGpp increased in parallel with increase of SpoT protein. The results indicate that polyamine stimulation of synthesis of SpoT and RpoZ plays important roles for cell viability through stimulation of ppGpp synthesis by SpoT and modulation of RNA synthesis by ppGpp-RpoZ complex.

  3. Narrow-Band Ultraviolet B Phototherapy Ameliorates Acute Graft-Versus-Host Disease of the Intestine by Expansion of Regulatory T Cells.

    Science.gov (United States)

    Hashimoto, Akari; Sato, Tsutomu; Iyama, Satoshi; Yoshida, Masahiro; Ibata, Soushi; Tatekoshi, Ayumi; Kamihara, Yusuke; Horiguchi, Hiroto; Murase, Kazuyuki; Kawano, Yutaka; Takada, Kohichi; Miyanishi, Koji; Kobune, Masayoshi; Ichimiya, Shingo; Kato, Junji

    2016-01-01

    Narrowband ultraviolet B (NB-UVB) has been widely used in dermatological phototherapy. As for the application of NB-UVB phototherapy to graft-versus-host disease (GVHD), we previously reported that it was highly efficacious for cutaneous lesions of acute GVHD (aGVHD) and that expansion of regulatory T (Treg) cells induced by NB-UVB might be one of the mechanisms. In order to examine whether NB-UVB irradiation through expansion of Treg cells is effective for the treatment of not only cutaneous aGVHD but also aGVHD of inner organs such as the intestine or liver, we conducted experiments in which a murine lethal aGVHD model, characterized by severe involvement of the intestine, was irradiated with NB-UVB. We found that NB-UVB irradiation improved the clinical score and survival rate. The pathological score of aGVHD was improved in all affected organs: intestine, liver, and skin. In the serum of mice irradiated with NB-UVB, the levels of Treg cells-associated cytokines such as transforming growth factor beta (TGFβ) and interleukin-10 (IL-10) were elevated. The numbers of infiltrating Treg cells in inflamed tissue of the intestine and those in spleen were increased in mice treated with NB-UVB. This is the first report demonstrating that NB-UVB phototherapy has the ability to ameliorate intestinal aGVHD through the expansion of Treg cells.

  4. Acute phase protein concentrations in serum and milk from healthy cows, cows with clinical mastitis and cows with extramammary inflammatory conditions

    NARCIS (Netherlands)

    Nielsen, B.H.; Jacobsen, S.; Andersen, P.H.; Niewold, T.A.; Heegaard, P.M.H.

    2004-01-01

    The concentrations of the two acute phase proteins, serum amyloid A and haptoglobin, in serum and milk were compared in 10 cows with clinical mastitis, 11 cows with extramammary inflammatory conditions and 10 clinically healthy control cows. The concentrations of both acute phase proteins were highe

  5. Lovastatin insensitive 1, a Novel pentatricopeptide repeat protein, is a potential regulatory factor of isoprenoid biosynthesis in Arabidopsis.

    Science.gov (United States)

    Kobayashi, Keiko; Suzuki, Masashi; Tang, Jianwei; Nagata, Noriko; Ohyama, Kiyoshi; Seki, Hikaru; Kiuchi, Reiko; Kaneko, Yasuko; Nakazawa, Miki; Matsui, Minami; Matsumoto, Shogo; Yoshida, Shigeo; Muranaka, Toshiya

    2007-02-01

    Higher plants have two metabolic pathways for isoprenoid biosynthesis: the cytosolic mevalonate (MVA) pathway and the plastidal non-mevalonate (MEP) pathway. Despite the compartmentalization of these two pathways, metabolic flow occurs between them. However, little is known about the mechanisms that regulate the two pathways and the metabolic cross-talk. To identify such regulatory mechanisms, we isolated and characterized the Arabidopsis T-DNA insertion mutant lovastatin insensitive 1 (loi1), which is resistant to lovastatin and clomazone, inhibitors of the MVA and MEP pathways, respectively. The accumulation of the major products of these pathways, i.e. sterols and chlorophyll, was less affected by lovastatin and clomazone, respectively, in loi1 than in the wild type. Furthermore, the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity analysis showed higher activity of HMGR in loi1-1 treated with lovastatin than that in the WT. We consider that the lovastatin-resistant phenotype of loi1-1 was derived from this post-transcriptional up-regulation of HMGR. The LOI1 gene encodes a novel pentatricopeptide repeat (PPR) protein. PPR proteins are thought to regulate the expression of genes encoded in organelle genomes by post-transcriptional regulation in mitochondria or plastids. Our results demonstrate that LOI1 is predicted to localize in mitochondria and has the ability to bind single-stranded nucleic acids. Our investigation revealed that the post-transcriptional regulation of mitochondrial RNA may be involved in isoprenoid biosynthesis in both the MVA and MEP pathways.

  6. Post-Translational Modifications of Kaposi’s Sarcoma-Associated Herpesvirus (KSHV Regulatory Proteins-SUMO and KSHV

    Directory of Open Access Journals (Sweden)

    Mel eCampbell

    2012-02-01

    Full Text Available Reactivation from a latent state is an important feature of infection and disease caused by many herpesviruses. KSHV latency can be envisioned as an outcome that is balanced between factors that promote viral gene expression and lytic replication against those that facilitate gene silencing and establish or maintain latency. A large body of work has focused on the activities of the key viral regulatory proteins involved in KSHV latent or lytic states. Moreover, recent studies have also begun to document the importance of epigenetic landscape evolution of the KSHV viral genome during latency and reactivation. However, one area of KSHV molecular virology that remains largely unanswered is the precise role of post-translational modifications on the activities of viral factors that function during latency and reactivation. In this review, we will summarize the post-translational modifications associated with three viral factors whose activities contribute to the viral state. The viral proteins discussed are the two major KSHV encoded transcription factors, K-Rta and K-bZIP (KSHV basic leucine zipper and the viral latency-associated nuclear antigen (LANA. A special emphasis will be placed on the role of the sumoylation pathway in the modulation of the KSHV lifecycle. Newly uncovered SUMO-dependent properties of LANA and K-Rta will also be presented, namely LANA histone-targeting SUMO E3 ligase activity and K-Rta SUMO-targeted ubiquitin ligase function.

  7. Regulation of steroid 5-{alpha} reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Young-Kyo [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States); Zhu, Bing [Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0144 (United States); Jeon, Tae-Il [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States); Osborne, Timothy F., E-mail: tfosborn@uci.edu [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States)

    2009-11-01

    In this study, we show that sterol regulatory element binding proteins (SREBPs) regulate expression of Srd5a2, an enzyme that catalyzes the irreversible conversion of testosterone to dihydroxytestosterone in the male reproductive tract and is highly expressed in androgen-sensitive tissues such as the prostate and skin. We show that Srd5a2 is induced in livers and prostate from mice fed a chow diet supplemented with lovastatin plus ezitimibe (L/E), which increases the activity of nuclear SREBP-2. The three fold increase in Srd5a2 mRNA mediated by L/E treatment was accompanied by the induction of SREBP-2 binding to the Srd5a2 promoter detected by a ChIP-chip assay in liver. We identified a SREBP-2 responsive region within the first 300 upstream bases of the mouse Srd5a2 promoter by co-transfection assays which contain a site that bound SREBP-2 in vitro by an EMSA. Srd5a2 protein was also induced in cells over-expressing SREBP-2 in culture. The induction of Srd5a2 through SREBP-2 provides a mechanistic explanation for why even though statin therapy is effective in reducing cholesterol levels in treating hypercholesterolemia it does not compromise androgen production in clinical studies.

  8. Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins.

    Directory of Open Access Journals (Sweden)

    Ayasa Ochiai

    Full Text Available Elevated plasma low-density lipoprotein (LDL cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD-15 cells, which lack insulin-induced gene-1 (Insig-1 and Insig-2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity.

  9. Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins.

    Science.gov (United States)

    Ochiai, Ayasa; Miyata, Shingo; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2015-01-01

    Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD-15 cells, which lack insulin-induced gene-1 (Insig-1) and Insig-2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity.

  10. Protein adsorption to monosodium urate crystals: differential responses of human peripheral blood neutrophils. [Etiology of acute gouty arthritis

    Energy Technology Data Exchange (ETDEWEB)

    Skosey, J.L.; Kozin, F.; Ginsberg, M.

    1976-01-01

    In order for acute gouty arthritis to occur, neutrophils must interact with monosodium urate (MSU) crystals. As a result of this interaction, enzymes, chemotactic factors, and other mediators of the inflammatory response are released from neutrophil lysosomes. It was observed that MSU crystals adsorb gamma globulin, albumin, and other proteins found in serum and joint fluid. Results are reported from a study designed to demonstrate the effects of coating of MSU crystals with proteins on the phlogistic responses of neutrophils to crystals.

  11. Preventing Phosphorylation of Sterol Regulatory Element-Binding Protein 1a by MAP-Kinases Protects Mice from Fatty Liver and Visceral Obesity

    OpenAIRE

    Jorg Kotzka; Birgit Knebel; Jutta Haas; Lorena Kremer; Sylvia Jacob; Sonja Hartwig; Ulrike Nitzgen; Dirk Muller-Wieland

    2012-01-01

    The transcription factor sterol regulatory element binding protein (SREBP)-1a plays a pivotal role in lipid metabolism. Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK). Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK) or p38 stress acti...

  12. Acute handling disturbance modulates plasma insulin-like growth factor binding proteins in rainbow trout (Oncorhynchus mykiss)

    Science.gov (United States)

    The effects of acute stressor exposure on proximal (growth hormone; GH) and distal (insulin-like growth factor-I; IGF-I and IGF-binding proteins) components of the somatotropic axis are poorly understood in finfish. We exposed rainbow trout (Oncorhynchus mykiss) to a 5-minute handling disturbance to...

  13. Effects of Acute Endurance Exercise on Plasma Protein Profiles of Endurance-Trained and Untrained Individuals over Time

    Directory of Open Access Journals (Sweden)

    Marius Schild

    2016-01-01

    Full Text Available Acute physical exercise and repeated exercise stimuli affect whole-body metabolic and immunologic homeostasis. The aim of this study was to determine plasma protein profiles of trained (EET, n=19 and untrained (SED, n=17 individuals at rest and in response to an acute bout of endurance exercise. Participants completed a bicycle exercise test at an intensity corresponding to 80% of their VO2max. Plasma samples were taken before, directly after, and three hours after exercise and analyzed using multiplex immunoassays. Seventy-eight plasma variables were included in the final analysis. Twenty-nine variables displayed significant acute exercise effects in both groups. Seven proteins differed between groups, without being affected by acute exercise. Among these A2Macro and IL-5 were higher in EET individuals while leptin showed elevated levels in SED individuals. Fifteen variables revealed group and time differences with elevated levels for IL-3, IL-7, IL-10, and TNFR2 in EET individuals. An interaction effect could be observed for nine variables including IL-6, MMP-2, MMP-3, and muscle damage markers. The proteins that differ between groups indicate a long-term exercise effect on plasma protein concentrations. These findings might be of importance in the development of exercise-based strategies in the prevention and therapy of chronic metabolic and inflammatory diseases and for training monitoring.

  14. Cytokine and acute phase protein gene expression in liver biopsies from dairy cows with a lipopolysaccharide - induced mastitis

    DEFF Research Database (Denmark)

    Vels, J; Røntved, Christine M.; Bjerring, Martin

    2009-01-01

    - ), IL-1β, IL-6, and IL-10, and the acute phase proteins serum amyloid A isoform 3 (SAA3), haptoglobin (Hp), and 1-acid glycoprotein (AGP) were determined by real-time reverse transcription-PCR. Fourteen primiparous cows in mid lactation were challenged with 200 µg of LPS (n = 8) or NaCl solution (n = 6...

  15. Increased Non-Enzymatic Glycation of Plasma Proteins and Hemoglobin in Non-Diabetic Patients with Acute Myocardial Infarction (MI)

    OpenAIRE

    Parthibane, V.; N. Selvaraj; Sathiyapriya, V.; Bobby, Zachariah; Rajappa, Medha

    2013-01-01

    Background: Glycation is known to play a key role in complications of many pathophysiological processes. The present study was carried out to assess whether there are abnormalities of non-enzymatic glycation of proteins and hemoglobin in acute Myocardial Infarction (MI) patients.

  16. Defective jejunal and colonic salt absorption and alteredNa +/H+ exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice

    NARCIS (Netherlands)

    N. Broere (Nellie); M. Chen (Min); A. Cinar (Ayhan); A.K. Singh (Arbind); J. Hillesheim (Jutta); B. Riederer (Beat Michel); M. Lunnemann; I. Rottinghaus (Ingrid); A. Krabbenhöft (Anja); R. Engelhardt (Regina); B. Rausch; E.J. Weinman (Edward); M. Donowitz (Mark); A. Hubbard; O. Kocher (Olivier); H.R. de Jonge (Hugo); B.M. Hogema (Boris); U. Seidler (Ursula)

    2009-01-01

    textabstractWe investigated the role of the Na+/H+ exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice display

  17. Regulatory Effect of Bcl-2 Family Proteins in CPB-induced Cardiomyocyte Apoptosis in Dog Hearts

    Institute of Scientific and Technical Information of China (English)

    孙宗全; 张顺业; 刘立新; 哈斯朝鲁

    2002-01-01

    Summary: Whether conventional hypothermic CPB induces myocyte apoptosis in dog hearts and modulation of bcl-2, bcl-xl, bax, bad, and caspase-3 pathways in this setting was investigated. Ten healthy adult dogs were randomized into sham-operated and CPB groups. Samples of left ventricle were obtained before, during and 3 h after CPB. In situ TUNEL was used to detect apoptotic my ocytes. Immunohistochemistry and flow cytometry were employed for detection of expressions of bcl 2, bcl-xl, bax and bad proteins. Z-DEVD-AMC substrate cleavage and TBARS methods were usedto measure the activity of caspase-3 and the content of lipid peroxide in LV myocardium, respective ly. After CPB, the number of apoptotic myocytes in CPB group was significantly increased. The re-sults of immunohistichemistry demonstrated that bcl-2, bcl-xl, bax and bad proteins were constitu tionally presnt on the sarcolemma of the LV myocytes. FACS results showed that, after CPB, ex-pressions of bax and bad in CPB group were significantly upregulated, while the expressions of bcl-2 and bcl-xl were not significantly changed in both groups. The activity of caspase-3 and the content of lipid peroxide in LV myocardium in CPB group were also significantly increased after CPB. The pre-sent study shows that there exists myocardiocyte apoptosis in dog hearts undergoing conventional hy-pothermic CPB and the myocyte apoptosis is initiated by ischemia and performed during reperfusion.Moreover, the CPB-induced myocyte apoptosis was associated with upregulation of expressions of bax and bad proteins, activation of caspase-3 and increase of oxidative stress.

  18. Regulatory effect of bcl-2 family proteins in CPB-induced cardiomyocyte apoptosis in dog hearts.

    Science.gov (United States)

    Sun, Zongquan; Zhang, Shunye; Liu, Lixin; Hasichaolu

    2002-01-01

    Whether conventional hypothermic CPB induces myocyte apoptosis in dog hearts and modulation of bcl-2, bcl-xl, bax, bad, and caspase-3 pathways in this setting was investigated. Ten healthy adult dogs were randomized into sham-operated and CPB groups. Samples of left ventricle were obtained before, during and 3 h after CPB. In situ TUNEL was used to detect apoptotic myocytes. Immunohistochemistry and flow cytometry were employed for detection of expressions of bcl-2, bcl-xl, bax and bad proteins. Z-DEVD-AMC substrate cleavage and TBARS methods were used to measure the activity of caspase-3 and the content of lipid peroxide in LV myocardium, respectively. After CPB, the number of apoptotic myocytes in CPB group was significantly increased. The results of immunohistochemistry demonstrated that bcl-2, bcl-xl, bax and bad proteins were constitutionally present on the sarcolemma of the LV myocytes. FACS results showed that, after CPB, expressions of bax and bad in CPB group were significantly upregulated, while the expressions of bcl-2 and bcl-xl were not significantly changed in both groups. The activity of caspase-3 and the content of lipid peroxide in LV myocardium in CPB group were also significantly increased after CPB. The present study shows that there exists myocardiocyte apoptosis in dog hearts undergoing conventional hypothermic CPB and the myocyte apoptosis is initiated by ischemia and performed during reperfusion. Moreover, the CPB-induced myocyte apoptosis was associated with upregulation of expressions of bax and bad proteins, activation of caspase-3 and increase of oxidative stress.

  19. Acute pancreatitis in aging animals: Loss of pancreatitis-associated protein protection?

    Institute of Scientific and Technical Information of China (English)

    Sophia Fu; Albert Stanek; Cathy M Mueller; Nefertti A Brown; Chongmin Huan; Martin H Bluth; Michael E Zenilman

    2012-01-01

    AIM:To investigate the effect of age on severity of acute pancreatitis (AP) using biochemical markers,histology and expression of the protective pancreatitisassociated proteins (PAPs).METHODS:AP was induced via intraductal injection of 4% sodium taurocholate in young and old rats.Sera and pancreata were assayed at 24 h for the parameters listed above; we also employed a novel molecular technique to assess bacterial infiltration using polymerase chain reaction to measure bacterial genomic ribosomal RNA.RESULTS:At 24 h after induction of AP,the pancreata of older animals had less edema (mean ± SE histologic score of young vs old:3.11 ± 0.16 vs 2.50 ±-0.11,P <0.05),decreased local inflammatory response (histologic score of stromal infiltrate:3.11 ± 0.27 vs 2.00 ± 0.17,P < 0.05) and increased bacterial infiltration (174% ±52% increase from sham vs 377% ± 4%,P < 0.05).A decreased expression of PAP1 and PAP2 was demonstrated by Western blotting analysis and immunohistochemical staining.There were no differences in serum amylase and lipase activity,or tissue myeloperoxidase or monocyte chemotactic protein-1 levels.However,in the most-aged group,serum C-reactive protein levels were higher (young vs old:0.249 ± 0.04 mg/dL vs 2.45± 0.68 mg/dL,P < 0.05).CONCLUSION:In older animals,there is depressed PAP expression related to a blunted inflammatory response in AP which is associated with worsened bacterial infiltration and higher C-reactive protein level; this may explain the more aggressive clinical course.

  20. Protein phosphatases and chromatin modifying complexes in the inflammatory cascade in acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Javier; Escobar; Javier; Pereda; Alessandro; Arduini; Juan; Sastre; Juan; Sandoval; Luis; Aparisi; Gerardo; López-Rodas; Luis; Sabater

    2010-01-01

    Acute pancreatitis is an inflammation of the pancreas that may lead to systemic inflammatory response syndrome and death due to multiple organ failure. Acinar cells, together with leukocytes, trigger the inflammatory cascade in response to local damage of the pancreas. Amplification of the inflammatory cascade requires up-regulation of proinflammatory cytokines and this process is mediated not only by nuclear factor κB but also by chromatinmodifying complexes and chromatin remodeling. Among the different families of histone acetyltransferases, the p300/CBP family seems to be particularly associated with the inflammatory process. cAMP activates gene expression via the cAMP-responsive element (CRE) and the transcription factor CRE-binding protein (CREB). CREB can be phosphorylated and activated by different kinases, such as protein kinase A and MAPK, and then it recruits the histone acetyltransferase co-activator CREB-binding protein (CBP) and its homologue p300. The recruitment of CBP/p300 and changes in the level of histone acetylation are required for transcription activation. Transcriptional repression is also a dynamic and essential mechanism of down-regulation of genes for resolution of inflammation, which seems to be mediated mainly by protein phosphatases (PP1, PP2A and MKP1) and histone deacetylases(HDACs) .Class HDACs are key transcriptional regulators whose activities are controlled via phosphorylationdependent nucleo/cytoplasmic shuttling. PP2A is responsible for dephosphorylation of class HDACs, triggeringnuclear localization and repression of target genes, whereas phosphorylation triggers cytoplasmic localization leading to activation of target genes. The potential benefit from treatment with phosphodiesterase inhibitors and histone deacetylase inhibitors is discussed.

  1. Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Larsen, Martin Røssel; Højrup, Peter;

    2004-01-01

    The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk...... by the modification of Thr213 but it does require the presence of an active Chk1 kinase....

  2. Oxytocin receptor gene sequences in owl monkeys and other primates show remarkable interspecific regulatory and protein coding variation.

    Science.gov (United States)

    Babb, Paul L; Fernandez-Duque, Eduardo; Schurr, Theodore G

    2015-10-01

    The oxytocin (OT) hormone pathway is involved in numerous physiological processes, and one of its receptor genes (OXTR) has been implicated in pair bonding behavior in mammalian lineages. This observation is important for understanding social monogamy in primates, which occurs in only a small subset of taxa, including Azara's owl monkey (Aotus azarae). To examine the potential relationship between social monogamy and OXTR variation, we sequenced its 5' regulatory (4936bp) and coding (1167bp) regions in 25 owl monkeys from the Argentinean Gran Chaco, and examined OXTR sequences from 1092 humans from the 1000 Genomes Project. We also assessed interspecific variation of OXTR in 25 primate and rodent species that represent a set of phylogenetically and behaviorally disparate taxa. Our analysis revealed substantial variation in the putative 5' regulatory region of OXTR, with marked structural differences across primate taxa, particularly for humans and chimpanzees, which exhibited unique patterns of large motifs of dinucleotide A+T repeats upstream of the OXTR 5' UTR. In addition, we observed a large number of amino acid substitutions in the OXTR CDS region among New World primate taxa that distinguish them from Old World primates. Furthermore, primate taxa traditionally defined as socially monogamous (e.g., gibbons, owl monkeys, titi monkeys, and saki monkeys) all exhibited different amino acid motifs for their respective OXTR protein coding sequences. These findings support the notion that monogamy has evolved independently in Old World and New World primates, and that it has done so through different molecular mechanisms, not exclusively through the oxytocin pathway.

  3. Identification and characterization of a novel regulatory factor: IgA-inducing protein.

    Science.gov (United States)

    Austin, Amy S; Haas, Karen M; Naugler, Sasha M; Bajer, Anna A; Garcia-Tapia, David; Estes, D Mark

    2003-08-01

    IgA is the predominant Ig isotype in mucosal secretions and thus plays a pivotal role in host defense. The mechanisms by which IgA expression is regulated may differ among species and involve multiple pathways. Various cytokines and costimulators have been identified which regulate expression of this isotype, including IL-10, IL-2, vasoactive intestinal peptide, and TGF-beta. We have tested a wide array of known factors, but only under very limited conditions do these factors mediate substantial IgA production in vitro from bovine B cells. In response to these findings, we generated a cDNA library in a mammalian expression vector from activated cells derived from bovine gut-associated lymphoid tissues (Peyer's patch and mesenteric lymph node cells) as a source of soluble factor(s) that may regulate IgA production. We have identified a novel factor, IgA-inducing protein, which stimulates relatively high levels of IgA production in vitro following CD40 stimulation in coculture with IL-2. Our data suggest that IgA-inducing protein regulates IgA by acting as a switch or differentiation factor and is expressed in a variety of lymphoid and nonlymphoid tissues.

  4. A novel Snf2 protein maintains trans-generational regulatory states established by paramutation in maize.

    Directory of Open Access Journals (Sweden)

    Christopher J Hale

    2007-10-01

    Full Text Available Paramutations represent heritable epigenetic alterations that cause departures from Mendelian inheritance. While the mechanism responsible is largely unknown, recent results in both mouse and maize suggest paramutations are correlated with RNA molecules capable of affecting changes in gene expression patterns. In maize, multiple required to maintain repression (rmr loci stabilize these paramutant states. Here we show rmr1 encodes a novel Snf2 protein that affects both small RNA accumulation and cytosine methylation of a proximal transposon fragment at the Pl1-Rhoades allele. However, these cytosine methylation differences do not define the various epigenetic states associated with paramutations. Pedigree analyses also show RMR1 does not mediate the allelic interactions that typically establish paramutations. Strikingly, our mutant analyses show that Pl1-Rhoades RNA transcript levels are altered independently of transcription rates, implicating a post-transcriptional level of RMR1 action. These results suggest the RNA component of maize paramutation maintains small heterochromatic-like domains that can affect, via the activity of a Snf2 protein, the stability of nascent transcripts from adjacent genes by way of a cotranscriptional repression process. These findings highlight a mechanism by which alleles of endogenous loci can acquire novel expression patterns that are meiotically transmissible.

  5. Novel functions for the endocytic regulatory proteins MICAL-L1 and EHD1 in mitosis.

    Science.gov (United States)

    Reinecke, James B; Katafiasz, Dawn; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like-1 (MICAL-L1) and C-terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL-L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi-nucleated cells. We provide evidence that bi-nucleation in MICAL-L1- and EHD1-depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL-L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1-independent function for MICAL-L1 earlier in mitosis. Moreover, we provide evidence that MICAL-L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL-L1 and EHD1 during the cell cycle.

  6. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    Science.gov (United States)

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian

    2016-07-01

    The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of peptide conformations. These findings are consistent with experimental results, and give a molecular level interpretation for the reduced cytotoxicity of Vpr13-33 to some extent upon graphene exposure. Meanwhile, this study provides some significant insights into the detailed mechanism of graphene-induced protein conformation transition.

  7. Structure and function of the regulatory HRDC domain from human Bloom syndrome protein.

    Science.gov (United States)

    Kim, Young Mee; Choi, Byong-Seok

    2010-11-01

    The helicase and RNaseD C-terminal (HRDC) domain, conserved among members of the RecQ helicase family, regulates helicase activity by virtue of variations in its surface residues. The HRDC domain of Bloom syndrome protein (BLM) is known as a critical determinant of the dissolution function of double Holliday junctions by the BLM-Topoisomerase IIIα complex. In this study, we determined the solution structure of the human BLM HRDC domain and characterized its DNA-binding activity. The BLM HRDC domain consists of five α-helices with a hydrophobic 3(10)-helical loop between helices 1 and 2 and an extended acidic surface comprising residues in helices 3-5. The BLM HRDC domain preferentially binds to ssDNA, though with a markedly low binding affinity (K(d) ∼100 μM). NMR chemical shift perturbation studies suggested that the critical DNA-binding residues of the BLM HRDC domain are located in the hydrophobic loop and the N-terminus of helix 2. Interestingly, the isolated BLM HRDC domain had quite different DNA-binding modes between ssDNA and Holliday junctions in electrophoretic mobility shift assay experiments. Based on its surface charge separation and DNA-binding properties, we suggest that the HRDC domain of BLM may be adapted for a unique function among RecQ helicases--that of bridging protein and DNA interactions.

  8. Social dominance-related major urinary proteins and the regulatory mechanism in mice.

    Science.gov (United States)

    Guo, Huifen; Fang, Qi; Huo, Ying; Zhang, Yaohua; Zhang, Jianxu

    2015-11-01

    Major urinary proteins (MUPs) have been proven to be non-volatile male pheromones in mice. Here, we aimed to elucidate the relationship between MUPs and dominance hierarchy, and the underlying molecular mechanisms. Dominance-submission relationship was established by chronic dyadic encountering. We found that at the urinary protein level and hepatic mRNA level, the expression of major MUPs, including Mup20, was enhanced in dominant males compared with subordinate males, indicating that MUPs might signal the social status of male mice. Meanwhile, the mRNA level of hepatic corticotropin releasing hormone receptor 2 (CRHR2) was higher in subordinate male mice than in dominant male mice. Castration also enhanced the expression of CRHR2, but suppressed that of MUPs. CRHR2 agonist treatment reduced the expression of MUPs in liver. However, male social status failed to exert significant influence on serum testosterone and corticosterone as well as the mRNA expression of their receptors. These findings reveal that some MUPs, especially Mup20, might constitute potential dominance pheromones and could be downregulated by hepatic CRHR2, which is possibly independent of androgen or corticosterone systems.

  9. Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Sato, Atsushi; Matsumura, Rie; Hoshino, Naomi; Tsuzuki, Mikio; Sato, Norihiro

    2014-01-01

    Triacylglycerol (TG) synthesis is induced for energy and carbon storage in algal cells under nitrogen(N)-starved conditions, and helps prevent reactive oxygen species (ROS) production through fatty acid synthesis that consumes excessive reducing power. Here, the regulatory mechanism for the TG content in sulfur(S)-starved cells of Chlamydomonas reinhardtii was examined, in comparison to that in N- or phosphorus(P)-starved cells. S- and N- starved cells exhibited markedly increased TG contents with up-regulation of mRNA levels of diacylglycerol acyltransferase (DGAT) genes. S-Starvation also induced expression of the genes for phosphatidate synthesis. In contrast, P-starved cells exhibited little alteration of the TG content with almost no induction of these genes. The results implied deficient nutrient-specific regulation of the TG content. An arg9 disruptant defective in arginine synthesis, even without nutritional deficiencies, exhibited an increased TG content upon removal of supplemented arginine, which repressed protein synthesis. Repression of protein synthesis thus seemed crucial for TG accumulation in S- or N- starved cells. Meanwhile, the results of inhibitor experiments involving cells inferred that TG accumulation during S-starvation is supported by photosynthesis and de novo fatty acid synthesis. During S-starvation, sac1 and snrk2.2 disruptants, which are defective in the response to the ambient S-status, accumulated TG at lower and higher levels, respectively, than the wild type. The sac1 and snrk2.2 disruptants showed no or much greater up-regulation of DGAT genes, respectively. In conclusion, TG synthesis would be activated in S-starved cells, through the diversion of metabolic carbon-flow from protein to TG synthesis, and simultaneously through up-regulation of the expression of a particular set of genes for TG synthesis at proper levels through the actions of SAC1 and SNRK2.2.

  10. Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Atsushi eSato

    2014-09-01

    Full Text Available Triacylglycerol (TG synthesis is induced for energy and carbon storage in algal cells under nitrogen(N-starved conditions, and helps prevent reactive oxygen species production through fatty acid synthesis that consumes excessive reducing power. Here, the regulatory mechanism for the TG content in sulfur(S-starved cells of Chlamydomonas reinhardtii was examined, in comparison to that in N- or phosphorus(P-starved cells. S- and N-starved cells exhibited markedly increased TG contents with up-regulation of mRNA levels of diacylglycerol acyltransferase genes. S-Starvation also induced expression of the genes for phosphatidate synthesis. In contrast, P-starved cells exhibited little alteration of the TG content with almost no induction of these genes. The results implied deficient nutrient-specific regulation of the TG content. An arg9 disruptant defective in arginine synthesis, even without nutritional deficiencies, exhibited an increased TG content upon removal of supplemented arginine, which repressed protein synthesis. Repression of protein synthesis thus seemed crucial for TG accumulation in S- or N-starved cells. Meanwhile, the results of inhibitor experiments involving cells inferred that TG accumulation during S-starvation is supported by photosynthesis and de novo fatty acid synthesis. During S-starvation, sac1 and snrk2.2 disruptants, which are defective in the response to the ambient S-status, accumulated TG at lower and higher levels, respectively, than the wild type. The sac1 and snrk2.2 disruptants showed no or much greater up-regulation of diacylglycerol acyltransferase genes, respectively. In conclusion, TG synthesis would be activated in S-starved cells, through the diversion of metabolic carbon-flow from protein to TG synthesis, and simultaneously through up-regulation of the expression of a particular set of genes for TG synthesis at proper levels through the actions of SAC1 and SNRK2.2.

  11. The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development.

    Science.gov (United States)

    Vivancos, Julien; Spinner, Lara; Mazubert, Christelle; Charlot, Florence; Paquet, Nicolas; Thareau, Vincent; Dron, Michel; Nogué, Fabien; Charon, Céline

    2012-03-01

    The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16-18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM.

  12. The flavone apigenin blocks nuclear translocation of sterol regulatory element-binding protein-2 in the hepatic cells WRL-68.

    Science.gov (United States)

    Wong, Tsz Yan; Lin, Shu-Mei; Leung, Lai K

    2015-06-28

    Sterol regulatory element-binding protein-2 (SREBP-2) is a pivotal transcriptional factor in cholesterol metabolism. Factors interfering with the proper functioning of SREBP-2 potentially alter plasma lipid concentrations. Consuming fruits and vegetables is associated with beneficial plasma lipid profile. The mechanism by which plant foods induce desirable lipid changes remains unclear. Apigenin, a common plant food flavonoid, was shown to modulate the nuclear translocation of SREBP-2 in the hepatic cells WRL-68 in the present study. The processing of SREBP-2 protein occurred after translation, and apigenin blocked this activation route. Further examination indicated that AMP-activated protein kinase (AMPK) was activated by the flavone, and co-administrating the AMPK-specific inhibitor compound C could release the blockage. Reporter gene assay revealed that the transactivation of sterol responsive element (SRE)-containing 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) promoter was suppressed by the flavone. Similarly, electromobility shift assay result also demonstrated a reduced DNA-binding activity on the SRE domain under the same treatment. The reduced transactivity and DNA-binding activity could be attributed to a decreased amount of SREBP-2 translocating from cytosol to nucleus as depicted by confocal microscopy. Quantitative RT-PCR assay demonstrated that the transcription of HMGCR followed the same pattern of SREBP-2 translocation. In summary, the present study showed that apigenin prevented SREBP-2 translocation and reduced the downstream gene HMGCR transcription. The minimum effective dosage should be achievable in the form of functional food consumption or dietary supplementation.

  13. Calpain-mediated cleavage of collapsin response mediator protein-2 drives acute axonal degeneration

    Science.gov (United States)

    Zhang, Jian-Nan; Michel, Uwe; Lenz, Christof; Friedel, Caroline C.; Köster, Sarah; d’Hedouville, Zara; Tönges, Lars; Urlaub, Henning; Bähr, Mathias; Lingor, Paul; Koch, Jan C.

    2016-01-01

    Axonal degeneration is a key initiating event in many neurological diseases. Focal lesions to axons result in a rapid disintegration of the perilesional axon by acute axonal degeneration (AAD) within several hours. However, the underlying molecular mechanisms of AAD are only incompletely understood. Here, we studied AAD in vivo through live-imaging of the rat optic nerve and in vitro in primary rat cortical neurons in microfluidic chambers. We found that calpain is activated early during AAD of the optic nerve and that calpain inhibition completely inhibits axonal fragmentation on the proximal side of the crush while it attenuates AAD on the distal side. A screening of calpain targets revealed that collapsin response mediator protein-2 (CRMP2) is a main downstream target of calpain activation in AAD. CRMP2-overexpression delayed bulb formation and rescued impairment of axonal mitochondrial transport after axotomy in vitro. In vivo, CRMP2-overexpression effectively protected the proximal axon from fragmentation within 6 hours after crush. Finally, a proteomic analysis of the optic nerve was performed at 6 hours after crush, which identified further proteins regulated during AAD, including several interactors of CRMP2. These findings reveal CRMP2 as an important mediator of AAD and define it as a putative therapeutic target. PMID:27845394

  14. Activation of a promyelocytic leukemia-tumor protein 53 axis underlies acute promyelocytic leukemia cure.

    Science.gov (United States)

    Ablain, Julien; Rice, Kim; Soilihi, Hassane; de Reynies, Aurélien; Minucci, Saverio; de Thé, Hugues

    2014-02-01

    Acute promyelocytic leukemia (APL) is driven by the promyelocytic leukemia (PML)-retinoic acid receptor-α (PML-RARA) fusion protein, which interferes with nuclear receptor signaling and PML nuclear body (NB) assembly. APL is the only malignancy definitively cured by targeted therapies: retinoic acid (RA) and/or arsenic trioxide, which both trigger PML-RARA degradation through nonoverlapping pathways. Yet, the cellular and molecular determinants of treatment efficacy remain disputed. We demonstrate that a functional Pml-transformation-related protein 53 (Trp53) axis is required to eradicate leukemia-initiating cells in a mouse model of APL. Upon RA-induced PML-RARA degradation, normal Pml elicits NB reformation and induces a Trp53 response exhibiting features of senescence but not apoptosis, ultimately abrogating APL-initiating activity. Apart from triggering PML-RARA degradation, arsenic trioxide also targets normal PML to enhance NB reformation, which may explain its clinical potency, alone or with RA. This Pml-Trp53 checkpoint initiated by therapy-triggered NB restoration is specific for PML-RARA-driven APL, but not the RA-resistant promyelocytic leukemia zinc finger (PLZF)-RARA variant. Yet, as NB biogenesis is druggable, it could be therapeutically exploited in non-APL malignancies.

  15. Hydroxysafflor yellow A suppress oleic acid-induced acute lung injury via protein kinase A

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Chaoyun [School of Pharmaceutical Sciences, Binzhou Medical University, Yantai, Shandong 264003 (China); Huang, Qingxian [Department of Hepatobiliary Surgery, Yantai Yuhuangding Hospital, Yantai, Shandong 264000 (China); Wang, Chunhua; Zhu, Xiaoxi; Duan, Yunfeng; Yuan, Shuai [School of Pharmaceutical Sciences, Binzhou Medical University, Yantai, Shandong 264003 (China); Bai, Xianyong, E-mail: xybai2012@163.com [School of Pharmaceutical Sciences, Binzhou Medical University, Yantai, Shandong 264003 (China)

    2013-11-01

    Inflammation response and oxidative stress play important roles in acute lung injury (ALI). Activation of the cAMP/protein kinase A (PKA) signaling pathway may attenuate ALI by suppressing immune responses and inhibiting the generation of reactive oxygen species (ROS). Hydroxysafflor yellow A (HSYA) is a natural flavonoid compound that reduces oxidative stress and inflammatory cytokine-mediated damage. In this study, we examined whether HSYA could protect the lungs from oleic acid (OA)-induced injury, which was used to mimic ALI, and determined the role of the cAMP/PKA signaling pathway in this process. Arterial oxygen tension (PaO{sub 2}), carbon dioxide tension, pH, and the PaO{sub 2}/fraction of inspired oxygen ratio in the blood were detected using a blood gas analyzer. We measured wet/dry lung weight ratio and evaluated tissue morphology. The protein and inflammatory cytokine levels in the bronchoalveolar lavage fluid and serum were determined using enzyme-linked immunoassay. The activities of superoxide dismutase, glutathione peroxidase, PKA, and nicotinamide adenine dinucleotide phosphate oxidase, and the concentrations of cAMP and malondialdehyde in the lung tissue were detected using assay kits. Bcl-2, Bax, caspase 3, and p22{sup phox} levels in the lung tissue were analyzed using Western blotting. OA increased the inflammatory cytokine and ROS levels and caused lung dysfunction by decreasing cAMP synthesis, inhibiting PKA activity, stimulating caspase 3, and reducing the Bcl-2/Bax ratio. H-89 increased these effects. HSYA significantly increased the activities of antioxidant enzymes, inhibited the inflammatory response via cAMP/PKA pathway activation, and attenuated OA-induced lung injury. Our results show that the cAMP/PKA signaling pathway is required for the protective effect of HSYA against ALI. - Highlights: • Oleic acid (OA) cause acute lung injury (ALI) via inhibiting cAMP/PKA signal pathway. • Blocking protein kinase A (PKA) activation may

  16. Regulatory roles of tumor necrosis factor alpha-induced proteins (TNFAIPs) 3 and 9 in arthritis.

    Science.gov (United States)

    Matsumoto, Isao; Inoue, Asuka; Takai, Chinatsu; Umeda, Naoto; Tanaka, Yuki; Kurashima, Yuko; Sumida, Takayuki

    2014-07-01

    Tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) have proved to be important in rheumatoid arthritis (RA) because the outcome of RA has greatly improved with the recent availability of biologics targeting them. It is well accepted that these cytokines are involved in the activation of the nuclear factor-κB (NF-κB) signaling pathway, but our understanding of the dependency of these pro-inflammatory cytokines and the link between them in RA is currently limited. Recently, we and others proved the importance of TNFα-induced protein (TNFAIP), due to the spontaneous development of arthritis in deficient animals that are dependent on IL-6. To date, nine TNFAIPs have been identified, and TNFAIP3 and TNFAIP9 were found to be clearly associated with mouse and human arthritis. In this review, we compare and discuss recent TNFAIP topics, especially focusing on TNFAIP3 and TNFAIP9 in autoimmune arthritis in mice and humans.

  17. Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein

    Science.gov (United States)

    Suzuki, Takashi; Brown, Judy J.; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5’-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5’-UTR for MTP-A. We generated reporter constructs in which the 5’-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5’-UTR, but not by the MTP-A 5’-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5’-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity. PMID:26771188

  18. Adaptive and maladaptive expression of the mRNA regulatory protein HuR

    Institute of Scientific and Technical Information of China (English)

    Suman; Govindaraju; Beth; S; Lee

    2013-01-01

    The RNA-binding proteins involved in regulation of mRNA post-transcriptional processing and translation control the fates of thousands of mRNA transcripts and basic cellular processes. The best studied of these, HuR, is well characterized as a mediator of mRNA stability and translation, and more recently, as a factor in nuclear functions such as pre-mRNA splicing. Due to HuR’s role in regulating thousands of mRNA transcripts, including those for other RNA-binding proteins, HuR can act as a master regulator of cell survival and proliferation. HuR itself is subject to multiple post-translationa modifications including regulation of its nucleocytoplasmic distribution. However, the mechanisms that govern HuR levels in the cell have only recently begun to be defined. These mechanisms are critical to cell health, as it has become clear in recent years that aberrant expression of HuR can lead alternately to decreased cell viability or to promotion of pathological proliferation and invasiveness. HuR is expressed as alternate mRNAs that vary in their untranslated regions, leading to differences in transcript stability and translatability. Multiple transcription factors and modulators of mRNA stability that regulate HuR mRNA expression have been identified. In addition, translation of HuR is regulated by numerous microRNAs, several of which have been demonstrated to have anti-tumor properties due to their suppression of HuR expression. This review summarizes the current state of knowledge of the factors that regulate HuR expression, along with the circumstances under which these factors contribute to cancer and inflammation.

  19. Effects of simvastatin on cardiac performance and expression of sarcoplasmic reticular calcium regulatory proteins in rat heart

    Institute of Scientific and Technical Information of China (English)

    Xia ZHENG; Shen-jiang HU

    2005-01-01

    Aim: To investigate the effect of simvastatin on the cardiac contractile function and the alteration of gene and protein expression of the sarcoplasmic calcium regulatory proteins, including sarcoplasmic reticulum Ca2+-ATPase (SERCA),phospholamban (PLB), and ryanodine receptor 2 (RyR2) in rat hearts. Methods:Langendorff-perfused rat hearts were subjected to 60-min perfusion with different concentrations of simvastatin (1, 3, 10, 30, or 100 μmol/L), and the parameters of cardiac function such as left ventricular developed pressure (LVDP), +dp/dtmax,and -dp/dtmax were determined. The cultured neonatal rat ventricular cardiomyocytes were incubated with simvastatin (1, 3, 10, 30, and 100 μmol/L) for 1 h or 24 h.The levels of SERCA, PLB, and RyR2 expression were measured by reverse transcription-polymerase chain reaction and Western blot. Cytotoxic effect of simvastatin on ventricular cardiomyocytes was assessed by the MTT colorimetric assay.Results: LVDP, +dp/dtmax, and -dp/dtmax of hearts were increased significantly after treatment with simvastatin 3, 10, and 30 μmol/L. In simvastatin-treated isolated hearts, the levels of mRNA expression of SERCA and RyR2 were elevated compared with the control (P<0.05), while the mRNA expression of PLB did not change. After the cultured neonatal rat ventricular cardiomyocytes were incubated with 3, 10, 30, and 100 μmol/L simvastatin for 1 h, SERCA and RyR2 mRNA expressions of cardiomyocytes rose, but there was no alteration in protein expressions. However, with the elongation of simvastatin treatment to 24 h, the protein expression of SERCA and RyR2 were also elevated. Additionally,simvastatin (1-30 μmol/L) had no influence on cell viability of cultured cardiac myocytes, but simvastatin 100 μmol/L inhibited the cell viability. Conclusion:Simvastatin improved cardiac performance accompanied by the elevation of SERCA and RyR2 gene and protein expression.

  20. Comparison of acute versus convalescent stage high-sensitivity C-Reactive protein level in predicting clinical outcome after acute ischemic stroke and impact of erythropoietin

    Directory of Open Access Journals (Sweden)

    Yeh Kuo-Ho

    2012-01-01

    Full Text Available Abstract Background and Aim Currently, no data on the optimal time point after acute ischemic stroke (IS at which high-sensitivity C-reactive protein (hs-CRP level is most predictive of unfavorable outcome. We tested the hypothesis that hs-CRP levels during both acute (48 h after IS and convalescent (21 days after IS phases are equally important in predicting 90-day clinical outcome after acute IS. We further evaluated the impact of erythropoietin (EPO, an anti-inflammatory agent, on level of hs-CRP after acute IS. Methods Totally 160 patients were prospectively randomized to receive either EPO therapy (group 1, n = 80 (5,000 IU each time, subcutaneously at 48 h and 72 h after acute IS, or placebo (group 2, n = 80. Serum level of hs-CRP was determined using ELISA at 48 h and on day 21 after IS and once in 60 healthy volunteers. Results Serum level of hs-CRP was substantially higher in all patients with IS than in healthy controls at 48 h and day 21 after IS (all p 0.5. Multivariate analysis showed that hs-CRP levels (at 48 h and day 21 were independently predictive of 90-day major adverse neurological event (MANE (defined as recurrent stroke, NIHSS≥8, or death (all p Conclusion EPO therapy which was independently predictive of freedom from 90-day MANE did not alter the crucial role of hs-CRP levels measured at 48 h and 21-day in predicting unfavorable clinical outcome after IS.

  1. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian, E-mail: jianzhou@scut.edu.cn

    2016-07-30

    Graphical abstract: Preferential adsorption of Vpr13-33 on graphene accompanied by early conformational change from α-helix to β-sheet structures was observed by molecular simulations. This work presents the molecular mechanism of graphene-induced peptide conformational alteration and sheds light on developing graphene-based materials to inhibit HIV. - Highlights: • Graphene induced early structural transition of Vpr13-33 is studied by MD simulations. • Both π-π stacking and hydrophobic interactions orchestrate the peptide adsorption. • Vpr has an increased propensity of β-sheet content on graphene surface. • To develop graphene-based materials to inhibit HIV is possible. - Abstract: The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of

  2. PLASMA C-REACTIVE PROTEIN LEVELS AS A PROGNOSTIC MARKER IN FIRST EVER ACUTE ISCHEMIC STROKE

    Directory of Open Access Journals (Sweden)

    Bharat

    2014-12-01

    Full Text Available BACKGROUND AND PURPOSE: Acute ischemic stroke may trigger an inflammatory response that leads to increased levels of C-reactive protein (CRP. High levels of CRP may be associated with poor outcome because they reflect either an inflammatory reaction or tissue damage. We related plasma CRP levels to first ever ischemic stroke and its role as a diagnostic aid. METHODS: Sixty patients fulfilling inclusion and exclusion criteria with first ever acute ischemic stroke were included in study. CT scan of brain was done after 24 hours of onset of symptoms to confirm the diagnosis. Plasma CRP level was determined after 12 hours and before 72 hours of onset of symptoms in all CT confirmed ischemic stroke patients. This clinical study was done from January 2008 to June 2009. CRP was randomly measured in 60 age and sex matched individuals admitted in other wards of the hospital matched in all possible criteria expect the disease under study as a control group. RESULTS: The CRP concentration in ischemic strokes was independent of infarction site, the value was more between 51-70 years of age group and almost equal in both genders. 54 of the 60 ischemic strokes studied had CRP value >6 mg/l and only 6 patients had 6 mg/l, which is insignificant. CONCLUSION: The CRP level is significantly higher in ischemic strokes and by its elevation between 12-72 hours of symptom onset is a bad prognostic indicator. The risk of poor outcome or death at 3 months increased with higher levels of CRP. Elevated CRP values is a risk factor in association with other risk factors like diabetes/hypertension

  3. The role of the acute phase protein PTX3 in the ventilator-induced lung injury

    Directory of Open Access Journals (Sweden)

    JM Real

    2008-06-01

    Full Text Available The pentraxin 3 (PTX3 is an acute phase proinflammatory protein produced by fibroblasts and alveolar epithelial cells. We have previously demonstrated that PTX3 is a key modulator of inflammation. Mechanical ventilation (MV is a life saving therapeutic approach for patients with acute lung injury that, nevertheless could lead to an inflammatory response and tissue injury (ventilator-induced lung injury: VILI, representing a major cause of iatrogenic lung damage in intensive units. Our objective was to investigate the role of PTX3 in VILI. PTX3 transgenic, knockout and Wt control mice (n = 12/group were ventilated (45ml·kg–1 until respiratory system Elastance increased 50% (Ers150%, an indicator of VILI. Histological analysis demonstrated that using a Ers150% was appropriate for our analysis since identical degrees of inflammation were observed in Tg, KO and Wt mice as assessed by leukocyte infiltration, oedema, alveolar collapse and number of breaks in alveolar septa. However, Tg mice reached Ers150% faster than Wt controls (p = 0.0225. We also showed that the lack of PTX3 does not abolish the occurrence of VILI in KOs. Gene expression profile of PTX3, IL-1beta, IL-6, KC, IFNgamma, TGFbeta and PCIII were investigated by QPCR. MV drastically up modulated PTX3 as well as IL-1beta, IL-6, IFNgamma and KC. Alternatively, mice were ventilated for 20, 40 and 60 min. The faster kinetics of Tg mice to reach Ers150% was accompanied by an earlier augmentation of IL-1b and PTX3 expression. The kinetics of local PTX3 expression in the lungs of ventilated mice strongly suggests the involvement of this pentraxin in the pathogenesis of VILI.

  4. ACUTE PHASE PROTEINS, LIPID PROFILE AND PROINFLAMMATORY CYTOKINES IN HEALTHY AND BRONCHOPNEUMONIC WATER BUFFALO CALVES

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    Sabry M. El-Bahr

    2013-01-01

    Full Text Available The aim of the present study was to evaluate the diagnostic value of Acute Phase Proteins (APP, lipid profiles and proinflammatory cytokines in healthy and bronchopneumonic water buffalo calves. Therefore, sixty water buffalo calves (9±1 month old, 175±15 kg were divided into two equal groups, the first group represented healthy, control, calves whereas calves of the second group were affected with bronchopneumonia. Total leukocytic and differential counts were determined. Serum total protein, albumin, Triacylglyceol (TAG, low density lipoprotein cholesterol (LDL-c, High Density Lipoprotein cholesterol (HDL-c, Total cholesterol, Alanine Amino Transferase (ALT, Aspartate Amino Transferase (AST, Alkaline Phosphatase (ALP, Fibrinogen (Fb, Haptaglobin (Hp, Serum Amyloid A (SAA, Tumor Necrosis Factor-alpha (TNF-α, Interleukins (IL1β, IL-12 and Interferon-gamma (IFN-γ were also determined. In addition, Bronchoalveolar Lavage (BAL was collected and analyzed. The present findings indicated that, total leukocytic and neutrophils counts were significantly (p<0.05 higher in pneumonic water buffalo calves compare with control. The examined biochemical parameters were significantly (p<0.05 increased in pneumonic calves except for total protein, albumin, cholesterol and HDL-c which were significantly (p<0.05 lower compare with control. Serum concentrations of investigated APP and proinflammatory cytokines were significantly (p<0.05 higher in pneumonic water buffalo calves than those of control. The present study demonstrated that, APP, lipid profile and proinflammatory cytokines perhaps served as biomarkers of bronchopneumonia in water buffalo calves. However, future studies with higher baseline sampling are still needed to establish and validate reference values for APP and cytokines in water buffalo calves.

  5. Renal and urinary levels of endothelial protein C receptor correlate with acute renal allograft rejection.

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    Lionel Lattenist

    Full Text Available The Endothelial Protein C Receptor (EPCR is expressed on leukocytes, on endothelium of large blood vessels and to a lesser extent on capillaries. Membrane bound EPCR plays an important role in the activation of protein C which has anticoagulant, anti-inflammatory and cytoprotective effects. After cleavage by a protease EPCR is also found as a soluble protein. Acute rejection of kidney allografts can be divided in T-cell-mediated rejection (TCMR and antibody-mediated (ABMR rejection. The latter is characterized by strong activation of coagulation. Currently no reliable non-invasive biomarkers are available to monitor rejection. Renal biopsies were available from 81 renal transplant patients (33 without rejection, 26 TCMR and 22 ABMR, we had access to mRNA material, matched plasma and urine samples for a portion of this cohort. Renal EPCR expression was assessed by RT-PCR and immunostaining. Plasma and urine sEPCR levels were measured by ELISA. ABMR patients showed higher levels of EPCR mRNA than TCMR patients. EPCR expression on glomeruli was significantly elevated in ABMR patients than in TCMR or control patients. In the peritubular capillaries EPCR expression was higher in ABMR patients than in control patients. EPCR expression was higher in tubules and arteries of rejection patients than in control patients. Plasma sEPCR levels did not differ. Urine sEPCR levels were more elevated in the ABMR group than in patients with TCMR or without rejection. ROC analysis demonstrated that urinary sEPCR is appropriate to discriminate between ABMR patients and TCMR or control patients. We conclude that urinary sEPCR could be a novel non-invasive biomarker of antibody mediated rejection in renal transplantation.

  6. Acute regulation of cardiac metabolism by the hexosamine biosynthesis pathway and protein O-GlcNAcylation.

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    Boglárka Laczy

    Full Text Available OBJECTIVE: The hexosamine biosynthesis pathway (HBP flux and protein O-linked N-acetyl-glucosamine (O-GlcNAc levels have been implicated in mediating the adverse effects of diabetes in the cardiovascular system. Activation of these pathways with glucosamine has been shown to mimic some of the diabetes-induced functional and structural changes in the heart; however, the effect on cardiac metabolism is not known. Therefore, the primary goal of this study was to determine the effects of glucosamine on cardiac substrate utilization. METHODS: Isolated rat hearts were perfused with glucosamine (0-10 mM to increase HBP flux under normoxic conditions. Metabolic fluxes were determined by (13C-NMR isotopomer analysis; UDP-GlcNAc a precursor of O-GlcNAc synthesis was assessed by HPLC and immunoblot analysis was used to determine O-GlcNAc levels, phospho- and total levels of AMPK and ACC, and membrane levels of FAT/CD36. RESULTS: Glucosamine caused a dose dependent increase in both UDP-GlcNAc and O-GlcNAc levels, which was associated with a significant increase in palmitate oxidation with a concomitant decrease in lactate and pyruvate oxidation. There was no effect of glucosamine on AMPK or ACC phosphorylation; however, membrane levels of the fatty acid transport protein FAT/CD36 were increased and preliminary studies suggest that FAT/CD36 is a potential target for O-GlcNAcylation. CONCLUSION/INTERPRETATION: These data demonstrate that acute modulation of HBP and protein O-GlcNAcylation in the heart stimulates fatty acid oxidation, possibly by increasing plasma membrane levels of FAT/CD36, raising the intriguing possibility that the HBP and O-GlcNAc turnover represent a novel, glucose dependent mechanism for regulating cardiac metabolism.

  7. Lipopolysaccharide binding protein and serum amyloid A secretion by human intestinal epithelial cells during the acute phase response.

    Science.gov (United States)

    Vreugdenhil, A C; Dentener, M A; Snoek, A M; Greve, J W; Buurman, W A

    1999-09-01

    The acute phase proteins LPS binding protein (LBP) and serum amyloid A (SAA) are produced by the liver and are present in the circulation. Both proteins have been shown to participate in the immune response to endotoxins. The intestinal mucosa forms a large surface that is continuously exposed to these microbial products. By secretion of antimicrobial and immunomodulating agents, the intestinal epithelium contributes to the defense against bacteria and their products. The aim of this study was to explore the influence of the inflammatory mediators TNF-alpha, IL-6, and IL-1beta on the release of LBP and SAA by intestinal epithelial cells (IEC). In addition, the induction of LBP and SAA release by cell lines of intestinal epithelial cells and hepatic cells was compared. The data obtained show that in addition to liver cells, IEC also expressed LBP mRNA and released bioactive LBP and SAA upon stimulation. Regulation of LBP and SAA release by IEC and hepatocytes was typical for class 1 acute phase proteins, although differences in regulation between the cell types were observed. Endotoxin did not induce LBP and SAA release. Glucocorticoids were demonstrated to strongly enhance the cytokine-induced release of LBP and SAA by IEC, corresponding to hepatocytes. The data from this study, which imply that human IEC can produce LBP and SAA, suggest a role for these proteins in the local defense mechanism of the gut to endotoxin. Furthermore, the results demonstrate that tissues other than the liver are involved in the acute phase response.

  8. Evidence that regulatory protein MarA of Escherichia coli represses rob by steric hindrance.

    Science.gov (United States)

    McMurry, Laura M; Levy, Stuart B

    2010-08-01

    The MarA protein of Escherichia coli can both activate and repress the initiation of transcription, depending on the position and orientation of its degenerate 20-bp binding site ("marbox") at the promoter. For all three known repressed genes, the marbox overlaps the promoter. It has been reported that MarA represses the rob promoter via an RNA polymerase (RNAP)-DNA-MarA ternary complex. Under similar conditions, we found a ternary complex for the repressed purA promoter also. These findings, together with the backwards orientation of repressed marboxes, suggested a unique interaction of MarA with RNAP in repression. However, no repression-specific residues of MarA could be found among 38 single-alanine replacement mutations previously shown to retain activation function or among mutants from random mutagenesis. Mutations Thr12Ala, Arg36Ala, Thr95Ile, and Pro106Ala were more damaging for activation than for repression, some up to 10-fold, so these residues may play a specific role in activation. We found that nonspecific binding of RNAP to promoterless regions of DNA was presumably responsible for the ternary complexes seen previously. When RNAP binding was promoter specific, MarA reduced RNAP access to the rob promoter; there was little or no ternary complex. These findings strongly implicate steric hindrance as the mechanism of repression of rob by MarA.

  9. Regulatory roles of tumor-suppressor proteins and noncoding RNA in cancer and normal cell functions.

    Science.gov (United States)

    Garen, Alan; Song, Xu

    2008-04-15

    We describe a mechanism for reversible regulation of gene transcription, mediated by a family of tumor-suppressor proteins (TSP) containing a DNA-binding domain (DBD) that binds to a gene and represses transcription, and RNA-binding domains (RBDs) that bind RNA, usually a noncoding RNA (ncRNA), forming a TSP/RNA complex that releases the TSP from a gene and reverses repression. This mechanism appears to be involved in the regulation of embryogenesis, oncogenesis, and steroidogenesis. Embryonic cells express high levels of RNA that bind to a TSP and prevent repression of proto-oncogenes that drive cell proliferation. The level of the RNA subsequently decreases in most differentiating cells, enabling a TSP to repress proto-oncogenes and stop cell proliferation. Oncogenesis can result when the level of the RNA fails to decrease in a proliferating cell or increases in a differentiated cell. This mechanism also regulates transcription of P450scc, the first gene in the steroidogenic pathway.

  10. An improved acute gouty arthritis rat model and therapeutic effect of rhizoma Dioscoreae nipponicae on acute gouty arthritis based on the protein-chip methods.

    Science.gov (United States)

    Yao, Li; Dong, Wanru; Lu, Fang; Liu, Shumin

    2012-01-01

    Rhizoma Dioscoreae nipponicae (RDN) is an herbal medicine. In the theories of Traditional Chinese Medicine (TCM), the function of RDN is to expel wind and remove dampness. Inflammatory mechanisms play an important role in the pathological process and prognosis of acute gouty arthritis (AGA). The aim of this study was to determine the specially expressed proteins through testing the proteins of the synovium in rats with AGA. The animal model of AGA was set up by Monosodium urate crystal (MSU) combined with hypoxanthine (HX), which was ameliorated in our previous experiment. Blood samples for measurement of serum uric acid were collected prior to sacrifice. Outcomes were assessed (two days after injection) by histological stain and protein quantitation. Three chips of RayBio® Human Label-based Antibody Array I were applied to detect 90 proteins in the synovium tissue of AGA rats. 14 differently expressed proteins were found in the synovium of AGA rats, and nine of them were first found in this model. There were seven up-regulated and seven down-regulated proteins, both TRAIL and Neuropilin-2 could be identified as key contributors to the pathomechanism of AGA.

  11. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada); Babiuk, Lorne A. [University of Alberta, Edmonton, Alberta (Canada); Liu, Qiang, E-mail: qiang.liu@usask.ca [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada)

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  12. Involvement of activated leukocytes in the regulation of plasma levels of acute phase proteins in microgravity simulation experiments

    Science.gov (United States)

    Larina, Olga; Bekker, Anna; Turin-Kuzmin, Alexey

    2016-07-01

    Earth-based studies of microgravity effects showed the induction of the mechanisms of acute phase reaction (APR). APR comprises the transition of stress-sensitive protein kinases of macrophages and other responsive cells into the active state and the phosphorylation of transcription factors which in turn stimulate the production of acute-phase reaction cytokines. Leukocyte activation is accompanied by the acceleration of the formation of oxygen radicals which can serve a functional indice of leukocyte cell state. The series of events at acute phase response result in selective changes in the synthesis of a number of secretory blood proteins (acute phase proteins, APPs) in liver cells thus contributing the recovery of homeostasis state in the organism. Earlier experiment with head-down tilt showed the increase in plasma concentrations of two cytokine mediators of acute phase response, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) being the outcome of the activation of producer cells, foremost, leukocytes. In experiment with 4-day dry immersion chemiluminescent (ChL) reply of the whole blood samples to a test stimulus were studied along with the measurements of plasma levels of APPs, namely, alpha1-antitrypsin (alpha1-AT), alpha1-acid glycoprotein (alpha1-AGP), alpha2-macroglobulin (alpha2-M), ceruloplasmin (Cer), haptoglobin (Hp), C3-complement component (C3), C-reactive protein (CRP). Eight individuals aged 21.2 ± 3.2 years were the test subjects in the investigation. Protein studies showed a noticeable increase in the mean plasma levels of all APPs measured in experiment thus producing the evidence of the activation of acute phase response mechanisms while individual patterns revealed variability during the immersion period. The overall trends were similar to these in the previous immersion series. The augment in the strength of signal in stimulated light emission tests was higher after 1- and 2-day of immersion exposure than before the

  13. A Model of Yeast Cell-Cycle Regulation Based on a Standard Component Modeling Strategy for Protein Regulatory Networks

    Science.gov (United States)

    Laomettachit, Teeraphan; Chen, Katherine C.; Baumann, William T.

    2016-01-01

    To understand the molecular mechanisms that regulate cell cycle progression in eukaryotes, a variety of mathematical modeling approaches have been employed, ranging from Boolean networks and differential equations to stochastic simulations. Each approach has its own characteristic strengths and weaknesses. In this paper, we propose a “standard component” modeling strategy that combines advantageous features of Boolean networks, differential equations and stochastic simulations in a framework that acknowledges the typical sorts of reactions found in protein regulatory networks. Applying this strategy to a comprehensive mechanism of the budding yeast cell cycle, we illustrate the potential value of standard component modeling. The deterministic version of our model reproduces the phenotypic properties of wild-type cells and of 125 mutant strains. The stochastic version of our model reproduces the cell-to-cell variability of wild-type cells and the partial viability of the CLB2-dbΔ clb5Δ mutant strain. Our simulations show that mathematical modeling with “standard components” can capture in quantitative detail many essential properties of cell cycle control in budding yeast. PMID:27187804

  14. Association of pro-inflammatory cytokines and iron regulatory protein 2 (IRP2 with Leishmania burden in canine visceral leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Paulo Ricardo Porfírio do Nascimento

    Full Text Available Leishmania infantum infection in humans and dogs can evolve with a wide range of clinical presentations, varying from asymptomatic infections to visceral leishmaniasis. We hypothesized that the immune response elicited by L. infantum infection could modulate whether the host will remain asymptomatic or progress to disease. A total of 44 dogs naturally infected with L. infantum were studied. Leishmania burden was estimated in the blood and spleen by qPCR. The expression of IFN-γ, TNF-α, IL-10 and Iron Regulatory Protein 2 (IRP2 were determined in the spleen by quantitative PCR. Sera cytokines were evaluated by ELISA. Dogs were grouped in quartiles according parasite burden. Increased expression of IFN-γ and TNF-α was associated with reduced Leishmania burden, whereas increased IL-10 and IRP2 expressions were associated with higher Leishmania load. Increased plasma albumin and IFN-γ expression explained 22.8% of the decrease in parasite burden in the spleen. These data confirm that lower IFN-γ response and higher IL-10 correlated with increased parasite load and severity of the visceral leishmaniasis in dogs. The balance between the branches of immune response and the intracellular iron availability could determine, in part, the course of Leishmania infection.

  15. Regulation of the CDP-choline pathway by sterol regulatory element binding proteins involves transcriptional and post-transcriptional mechanisms.

    Science.gov (United States)

    Ridgway, Neale D; Lagace, Thomas A

    2003-06-15

    The synthesis of phosphatidylcholine (PtdCho) by the CDP-choline pathway is under the control of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT). Sterol regulatory element binding proteins (SREBPs) have been proposed to regulate CCT at the transcriptional level, or via the synthesis of lipid activators or substrates of the CDP-choline pathway. To assess the contributions of these two mechanisms, we examined CCTalpha expression and PtdCho synthesis by the CDP-choline pathway in cholesterol and fatty acid auxotrophic CHO M19 cells inducibly expressing constitutively active nuclear forms of SREBP1a or SREBP2. Induction of either SREBP resulted in increased expression of mRNAs for sterol-regulated genes, elevated fatty acid and cholesterol synthesis (>10-50-fold) and increased PtdCho synthesis (2-fold). CCTalpha mRNA was increased 2-fold by enforced expression of SREBP1a or SREBP2. The resultant increase in CCTalpha protein and activity (2-fold) was restricted primarily to the soluble fraction of cells, and increased CCTalpha activity in vivo was not detected. Inhibition of the synthesis of fatty acids or their CoA esters by cerulenin or triacsin C respectively following SREBP induction effectively blocked the accompanying elevation in PtdCho synthesis. Thus PtdCho synthesis was driven by increased synthesis of fatty acids or a product thereof. These data show that transcriptional activation of CCTalpha is modest relative to that of other SREBP-regulated genes, and that stimulation of PtdCho synthesis by SREBPs in CHO cells is due primarily to increased fatty acid synthesis.

  16. Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.

    Science.gov (United States)

    Peretti, Leandro E; Gonzalez, Verónica D G; Marcipar, Iván S; Gugliotta, Luis M

    2014-08-01

    The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease.

  17. Phenotypes and clinical significance of circulating CD4+CD25+ regulatory T cells (Tregs in patients with acute-on-chronic liver failure (ACLF

    Directory of Open Access Journals (Sweden)

    Yang Jiezuan

    2012-09-01

    Full Text Available Abstract Background CD4+CD25+ regulatory T cells (Tregs play an important role in maintaining immunological tolerance to self and foreign antigens. T cell receptors (TCR reflect the composition and function of T cells. It is not universally agreed that there is a relationship between CD4+CD25+ Treg frequency and the severity of acute-on-chronic liver failure (ACLF. The repertoire of TCR beta chain variable (TCRBV regions of peripheral Tregs in ACLF patients is not well understood. Methods Human PBMCs were separated and sorted into CD4+CD25+ Treg subsets using density gradient centrifugation and magnetic activated cell sorting (MACS. The CD4+CD25high Treg frequency in peripheral blood of ACLF and chronic hepatitis B (CHB patients was measured by flow cytometry. The molecular profiles of TCRBV CDR3 were determined using gene melting spectral pattern (GMSP analysis. TCRBV gene families were cloned and sequenced when the GMSP profiles showed a single-peak. Results CD4+CD25high Treg prevalence in peripheral blood of ACLF patients is increased significantly compared to healthy donors (HDs (P P +CD25high Tregs in ACLF or CHB patients is positively correlated with HBV DNA load. The TCRBV11, BV13.1, BV18, BV20 are the most prevalent TCRBV in CD4+CD25+ Tregs in ACLF and CHB patients. In addition, the CDR3 motifs were relatively conserved in these four TCRBV gene families. Conclusions The CD4+CD25high Tregs prevalence in peripheral blood is indicative of disease severity in ACLF or CHB patients. The relatively conserved TCRBV20 CDR3 motif “TGTGHSPLH” and TCRBV11 CDR3 motif “VYNEQ” may be used in helping diagnosis and treat patients with ACLF.

  18. Acute Exacerbations of Chronic Hepatitis B Are Accompanied by Decline of Core Antigen-Specific Regulatory T-Cell Frequencies: Implications for Successful Anti-HBV Treatments

    Directory of Open Access Journals (Sweden)

    Sun-Lung Tsai

    2007-11-01

    Full Text Available Background and Aims: Acute exacerbations (AEs of perinatally-acquired chronic hepatitis B (CHB are accompanied by increased T cell responses to hepatitis B core and e antigens (HBcAg & HBeAg. Naturally-arising forkhead transcription factor Foxp3 (forkhead box p3-expressing CD4+CD25+ regulatory T (Treg cells are thought to be important in the control of infectious diseases. This study aimed to investigate whether HBcAg-specific Treg cells play a role in modulating spontaneous AEs and in influencing the outcome of anti-hepatitis B virus (HBV treatments.Methods: The SYFPEITHI scoring system was employed to predict epitope peptides on HBcAg overlapping with HBeAg for the construction of peptide-HLA class II tetramers to measure HBcAg-specific Treg cell frequencies (Treg f . Results: HBcAg-specific Treg f declined significantly in association with increased HBcAg-specific cytotoxic T lymphocyte frequencies during spontaneous AEs without treatment. Vigorous in vitro expansion of CD4+CD25+ Treg cells from CHB patients responding to HBcAg and/or its peptides plus interleukin-2 (IL-2 was consistently detected. Depletion of Treg cells from peripheral blood mononuclear cells enhanced proliferation to HBcAg. In contrast, patients with AEs who received anti-HBV treatments with oral nucleoside analogues or interferon-alpha injection revealed that more post-treatment increase of HBcAg-specific Treg f correlated with a higher sustained remission rate to the therapy.Conclusions: These data indicate that HBcAg-specific Treg cells from perinatally-acquired CHB patients are proliferative to HBcAg and its peptides and exhibit suppressor activity. They play a crucial role in modulating spontaneous AEs and in successful anti-HBV treatments.

  19. Pig α1-Acid Glycoprotein: Characterization and First Description in Any Species as a Negative Acute Phase Protein

    DEFF Research Database (Denmark)

    Heegaard, Peter M. H.; Miller, Ingrid; Sørensen, Nanna Skall;

    2013-01-01

    concentrations of pig AGP were in the range of 1-3 mg/ml in a number of conventional pig breeds while it was lower in Göttingen and Ossabaw minipigs (in the 0.3 to 0.6 mg/ml range) and higher in young (2-5 days old) conventional pigs (mean: 6.6 mg/ml). Surprisingly, pig AGP was found to behave as a negative...... acute phase protein during a range of experimental infections and aseptic inflammation with significant decreases in serum concentration and in hepatic ORM1 expression during the acute phase response. To our knowledge this is the first description in any species of AGP being a negative acute phase...

  20. Erythrocyte heat shock protein responses to chronic (in vivo) and acute (in vitro) temperature challenge in diploid and triploid salmonids.

    Science.gov (United States)

    Saranyan, Pillai V; Ross, Neil W; Benfey, Tillmann J

    2017-04-01

    This research investigated how ploidy level (diploid versus triploid) affects the heat shock protein (HSP) response in erythrocytes under different thermal stress regimes, both in vivo and in vitro, in Atlantic salmon (Salmo salar) and brook charr (Salvelinus fontinalis) in order to address the question of why triploids typically have reduced thermal tolerance. A preliminary study confirmed that identical volumes of diploid and triploid erythrocytes (which equates to a smaller number of larger cells for triploids compared to diploids) did not differ in total protein synthesis rates. After chronic (100d) acclimation of fish to 5, 15 and 25°C, triploid erythrocytes had lower HSP70, HSP90, heat shock factor 1 (HSF1) and ubiquitin (free and total) levels than diploids in both species. Furthermore, Atlantic salmon erythrocytes showed significantly higher protein breakdown (based on conjugated ubiquitin levels) in triploids than diploids after acute heat stress in vitro, but no significant difference was detected between ploidies after acute cold stress. These results indicate that: 1) triploid erythrocytes synthesize more total protein per cell than diploids as a result of increased cell size; 2) triploids have sufficient total HSP levels for survival under low stress conditions; and 3) the lower basal titres of HSPs in triploids may be a handicap when combating acute stress. Taken together, this suggests that triploids are limited in their ability to withstand thermal stress because of a reduced ability to maintain proteostasis under stressful conditions.

  1. Serum acute phase proteins as biomarkers of pleuritis and cranio-ventral pulmonary consolidation in slaughter-aged pigs.

    Science.gov (United States)

    Saco, Yolanda; Fraile, Lorenzo; Giménez, Mercè; Alegre, Ana; López-Jimenez, Rosa; Cortey, Martí; Segalés, Joaquim; Bassols, Anna

    2011-08-01

    The purpose of this study was to investigate the relationship between the existence of lung lesions in pigs at slaughter and the concentration of the serum acute phase proteins (APP), haptoglobin (Hp), pig-major acute protein (Pig-MAP) and C-reactive protein (CRP). A total of 24 pig farms were selected out of a larger farm database previously screened to study risk factors associated with pleuritis and cranio-ventral pulmonary consolidation (CVPC) lesions at slaughter-aged pigs in Spain. The farms were classified as "pleuritis negative (P-) or positive (P+)" and as "CVPC negative (M-) or positive (M+)" and divided into four groups according to a 2X2 factorial design (P-M-, P-M+, P+M-, P+M+). Also at slaughter, blood from 20 randomly selected pigs from each farm was collected. Obtained serum samples were used to measure acute phase proteins. All APP concentrations were significantly higher for M+ farms than for M- ones. However, only Hp and Pig-MAP showed significantly higher concentrations for P+ farms than for P- ones. Pig-MAP was the most sensitive biomarker since it was able to clearly discriminate between P-/P+ and M-/M+ groups (ppleuritis and good for CVPC lesions. CRP was able to discriminate for CVPC lesions but not for pleuritis. The present results indicate that Pig-MAP and, possibly Hp, may be used as potential markers to characterise and discriminate respiratory lesions in swine herds at slaughter.

  2. Heat Shock Protein 72 Protects Retinal Ganglion Cells in Rat Model of Acute Glaucoma

    Institute of Scientific and Technical Information of China (English)

    Guoping Qing; Xuanchu Duan; Youqin Jiang

    2005-01-01

    Purpose: To investigate whether the induction of heat shock protein (HSP)72 by heat stress (HS) or zinc (Zn2+ ) administration can increase survival of retinal ganglion cells (RGC) in rat model of acute experimental glaucoma.Methods: Acute glaucoma model was made by intracameral irrigation with BSS at 102 mmHg for two hours in right eyes of male Wistar rats. Glaucoma model rats were treated with HS once a week (six rats) or intraperitoneal injection of zinc sulfate (24.6 mg/kg) every two weeks (six rats), and were referred to as HS group and zinc group, respectively. Untreated model rats served as damage group (six rats). In control groups, quercetin (400 mg/kg) was intraperitoneally injected to inhibit the induction of heat shock proteins 6 hours before HS or zinc administration, and were referred to as HS+que group (six rats) and zinc+que group (six rats), respectively. Subsequent to 16 days of IOP elevation, the rats were sacrificed. Eyes were quickly enucleated, and the retinas were dissected. RGC were labeled with Nissl staining and counted under microscope.Results: The average RGC density in normal Wistar rats was (2504±181) cells/mm2. In damage group, it decreased to (2015±111 ) cells/mm2. The RGC densities at 1,2, and 3 mm from the center of the optic nerve head were (2716±215), (2496±168), and (2317±171) cells/mm2, respectively, for normal rats and (2211±133), (1969±154),and (1872±68) cells/mm2, respectively, for damage group. The latter was significantly lower at all locations compared with the former (P=0.027 for each, Mann-Whitney test).The average RGC densities were (2207±200) cells/mm2 for HS group, (2272±155) cells/mm2 for zinc group, (1964±188) cells/mm2 for HS+que group, (2051 ±214) cells/mm2 for zinc+que group and (2015±111 ) cells/mm2 for damage group. There were significant differences in density of labeled RGCs among the five groups (P=0.040,Kruskal-Wallis test). Both HS and zinc group had higher RGC densities than damage group (P

  3. Antibody SPC-54 provides acute in vivo blockage of the murine protein C system.

    Science.gov (United States)

    Burnier, Laurent; Fernández, José A; Griffin, John H

    2013-04-01

    Multiple protective effects of pharmacological activated protein C (APC) are reported in several organ pathologies. To help evaluate the endogenous murine PC system, we characterized a rat monoclonal anti-mouse PC antibody, SPC-54, which inhibited the amidolytic and anticoagulant activities of murine APC by>95%. SPC-54 blocked active site titration of purified APC using the active site titrant, biotinylated FPR-chloromethylketone, showing that SPC-54 blocks access to APC's active site to inhibit all enzymatic activity. A single injection of SPC-54 (10mg/kg) neutralized circulating PC in mice for at least 7days, and immunoblotting and immuno-precipitation with protein G-agarose confirmed that SPC-54 in vivo was bound to PC in plasma. Pre-infusion of SPC-54 in tissue factor-induced murine acute thromboembolism experiments caused a major decrease in mean survival time compared to controls (7min vs. 42.5min, P=0.0016). SPC-54 decreased lung perfusion in this model by 54% when monitored by vascular perfusion methodologies using infrared fluorescence of Evans blue dye. In LD50 endotoxemia murine models, SPC-54 infused at 7hr after endotoxin administration increased mortality from 42% to 100% (PSPC-54 ablates in vitro and in vivo APC protective functions and enzymatic activity. The ability of SPC-54 to block the endogenous PC/APC system provides a powerful tool to understand better the role of the endogenous PC system in murine injury models and in cell bioassays and also to neutralize the enzymatic activities of murine APC in any assay system.

  4. Serum heat shock protein 60 in acute heart failure: a new biomarker?

    Science.gov (United States)

    Bonanad, Clara; Núñez, Julio; Sanchis, Juan; Bodi, Vicente; Chaustre, Fabian; Chillet, Marifina; Miñana, Gema; Forteza, Maria José; Palau, Patricia; Núñez, Eduardo; Navarro, David; Llàcer, Angel; Chorro, Francisco J

    2013-01-01

    Heat shock protein 60 (HSP60) is a mitochondrial protein constitutively expressed in the majority of cells, and its expression is up-regulated by a variety of stressors. In heart failure, HSP60 is released from cardiomyocytes. The authors speculate that increased serum HSP60 (sHSP60) may be related to the severity of heart failure. This investigation sought to assess the association between sHSP60 and the composite end point of death/readmission in patients with acute heart failure (AHF). A total of 132 consecutive patients were admitted for AHF. The independent association between sHSP60 and the end point was assessed with Cox regression. During a median follow-up of 7 months (interquartile range, 3-14), 35 (26.5%) deaths, 40 (30.3%) readmissions, and 65 (49.2%) deaths/readmission were identified. Patients who exhibited the outcome showed higher median sHSP60 values (6.15 ng/mL [8.49] vs 4.71 ng/mL [7.55] P=.010). A monotonic increase in the incidence of the composite end point was observed when moving from lower to higher tertile (4.74, 4.76, and 6.98 per 10 patients-years of follow-up, P for trend <.001). After adjusting for established risk factors, only patients in the upper tertile showed an increased risk of death/readmission (hazard ratio, 2.63; 95% confidence interval, 1.29-5.37; P=.008). In patients with AHF, high sHSP60 was related to a higher risk for subsequent death/readmission for AHF.

  5. Properties of the phage-shock-protein (Psp) regulatory complex that govern signal transduction and induction of the Psp response in Escherichia coli.

    Science.gov (United States)

    Jovanovic, Goran; Engl, Christoph; Mayhew, Antony J; Burrows, Patricia C; Buck, Martin

    2010-10-01

    The phage-shock-protein (Psp) response maintains the proton-motive force (pmf) under extracytoplasmic stress conditions that impair the inner membrane (IM) in bacterial cells. In Escherichia coli transcription of the pspABCDE and pspG genes requires activation of σ(54)-RNA polymerase by the enhancer-binding protein PspF. A regulatory network comprising PspF-A-C-B-ArcB controls psp expression. One key regulatory point is the negative control of PspF imposed by its binding to PspA. It has been proposed that under stress conditions, the IM-bound sensors PspB and PspC receive and transduce the signal(s) to PspA via protein-protein interactions, resulting in the release of the PspA-PspF inhibitory complex and the consequent induction of psp. In this work we demonstrate that PspB self-associates and interacts with PspC via putative IM regions. We present evidence suggesting that PspC has two topologies and that conserved residue G48 and the putative leucine zipper motif are determinants required for PspA interaction and signal transduction upon stress. We also establish that PspC directly interacts with the effector PspG, and show that PspG self-associates. These results are discussed in the context of formation and function of the Psp regulatory complex.

  6. Abnormal expression of FLI1 protein is an adverse prognostic factor in acute myeloid leukemia

    Science.gov (United States)

    Qiu, Yi Hua; Zhang, Nianxiang; Singh, Neera; Faderl, Stefan; Ferrajoli, Alessandra; York, Heather; Qutub, Amina A.; Coombes, Kevin R.; Watson, Dennis K.

    2011-01-01

    Friend leukemia virus integration 1 (FLI1), an Ets transcription factor family member, is linked to acute myelogenous leukemia (AML) by chromosomal events at the FLI1 locus, but the biologic impact of FLI1 expression on AML is unknown. FLI1 protein expression was measured in 511 newly diagnosed AML patients. Expression was similar in peripheral blood (PB) and BM and higher at diagnosis than at relapse (P = .02). Compared with normal CD34+ cells, expression in AML was above or below normal in 32% and 5% of patients, respectively. Levels were negatively correlated with an antecedent hematologic disorder (P = .002) but not with age or cytogenetics. Mutated NPM1 (P = .0007) or FLT3-ITD (P 0.3) with 19 others. The FLI1 level was not predictive of remission attainment, but patients with low or high FLI1 expression had shorter remission duration (22.6 and 40.3 vs 51.1 weeks, respectively; P = .01) and overall survival (45.2 and 35.4 vs 59.4 weeks, respectively; P = .03). High FLI1 levels were adverse in univariate and multivariate analysis. FLI1 expression is frequently abnormal and prognostically adverse in AML. FLI1 and/or its response genes may be therapeutically targetable to interfere with AML cell biology. PMID:21917756

  7. Effect of BCAA Supplementation on Serum C - Reactive Protein after Acute Resistance Exercise in Soccer Players

    Directory of Open Access Journals (Sweden)

    Mohammad Ghaderi

    2013-01-01

    Full Text Available Introduction: The present study was conducted to assess the effect of BCAA supplementation on serum C - reactive protein, after acute resistance exercise in soccer players.Methods: 20 soccer players in a randomized one-blind design completed in random 3 sets of 8-10 repetitions of 7 exercises. Subjects were randomized to two ten-member groups and consumed 200 mg. kg-1 BW of either BCAA or dextrin (placebo 30 minutes prior to exercise. To identify HS-CRP, venous blood samples were obtained 30 min prior to and immediately following exercise and at 1 hr, 2 hrs, 24hrs post exercise. Data were analyzed using 2-way repeated measure ANOVA and Bonferroni post hoc test. Results: Baseline serum values for Hs-CRP were not statistically different between groups in the 30 minutes before the exercise test (P>0.05. However there were significant increases (P<0.05 between the pre exercise and post exercise values for Hs-CRP from 24 hrs posttest, (P<0.05(. Importantly, the BCAA supplementation significantly reduced this Hs-CRP 24hrs post-test (P<0.05(.Conclusion: These results indicate that supplementary BCAA decreased serum concentrations of the inflammation biomarker Hs-CRP following resistance exercise. This observation suggests that BCAA supplementation may reduce the inflammation biomarker associated with resistance exercise.

  8. Severe acute respiratory syndrome coronavirus protein 6 mediates ubiquitin-dependent proteosomal degradation of N-Myc(and STAT) interactor

    Institute of Scientific and Technical Information of China (English)

    Weijia; Cheng; Shiyou; Chen; Ruiling; Li; Yu; Chen; Min; Wang; Deyin; Guo

    2015-01-01

    Severe acute respiratory syndrome coronavirus(SARS-Co V) encodes eight accessory proteins, the functions of which are not yet fully understood. SARS-Co V protein 6(P6) is one of the previously studied accessory proteins that have been documented to enhance viral replication and suppress host interferon(IFN) signaling pathways. Through yeast two-hybrid screening, we identified eight potential cellular P6-interacting proteins from a human spleen c DNA library. For further investigation, we targeted the IFN signaling pathway-mediating protein, N-Myc(and STAT) interactor(Nmi). Its interaction with P6 was confirmed within cells. The results showed that P6 can promote the ubiquitin-dependent proteosomal degradation of Nmi. This study revealed a new mechanism of SARS-Co V P6 in limiting the IFN signaling to promote SARS-Co V survival in host cells.

  9. Human herpesvirus 8 interferon regulatory factor-mediated BH3-only protein inhibition via Bid BH3-B mimicry.

    Directory of Open Access Journals (Sweden)

    Young Bong Choi

    Full Text Available Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of host cells. For HHV-8, viral interferon regulatory factor-1 (vIRF-1 contributes to this process in part via inhibitory interactions with BH3-only protein (BOP Bim, recently identified as an interaction partner of vIRF-1. Here we recognize that the Bim-binding domain (BBD of vIRF-1 resembles a region (BH3-B of Bid, another BOP, which interacts intramolecularly with the functional BH3 domain of Bid to inhibit it pro-apoptotic activity. Indeed, vIRF-1 was found to target Bid in addition to Bim and to interact, via its BBD region, with the BH3 domain of each. In functional assays, BBD could substitute for BH3-B in the context of Bid, to suppress Bid-induced apoptosis in a BH3-binding-dependent manner, and vIRF-1 was able to protect transfected cells from apoptosis induced by Bid. While vIRF-1 can mediate nuclear sequestration of Bim, this was not the case for Bid, and inhibition of Bid and Bim by vIRF-1 could occur independently of nuclear localization of the viral protein. Consistent with this finding, direct BBD-dependent inactivation by vIRF-1 of Bid-induced mitochondrial permeabilization was demonstrable in vitro and isolated BBD sequences were also active in this assay. In addition to Bim and Bid BH3 domains, BH3s of BOPs Bik, Bmf, Hrk, and Noxa also were found to bind BBD, while those of both pro- and anti-apoptotic multi-BH domain Bcl-2 proteins were not. Finally, the significance of Bid to virus replication was demonstrated via Bid-depletion in HHV-8 infected cells, which enhanced virus production. Together, our data demonstrate and characterize BH3 targeting and associated inhibition of BOP pro-apoptotic activity by vIRF-1 via Bid BH3-B mimicry, identifying a novel mechanism of viral evasion from host cell defenses.

  10. Decreased expression of complement regulatory proteins, CD55 and CD59, on peripheral blood leucocytes in patients with type 2 diabetes and macrovascular diseases

    Institute of Scientific and Technical Information of China (English)

    MA Xi-wen; CHANG Zhi-wen; QIN Ming-zhao; SUN Ying; HUANG Hui-lian; HE Yan

    2009-01-01

    Background Macro- and microvascular diseases are the leading cause of morbidity and mortality in diabetic patients, but their mechanisms remain unclear. Recent reports provide evidence that the levels of CD55 and CD59 are decreased in diabetic microvascular diseases. However, very little is known about the levels of CD55 and CD59, the relationship between them and carotid artery intima-media thickness, and the effects of statins on CD55 and CD59 in diabetic macrovascular diseases.Methods The mean fluorescence intensity (MFI) of CD55 and CD59 expression on peripheral blood leucocyte subsets (lymphocytes, monocytes and neutrophils) was studied using flow cytometry, and carotid artery intima-media thickness was measured using B-mode ultrasonography in 23 healthy subjects (controls), 19 patients with type 2 diabetes (T2DM), and 43 patients with type 2 diabetes and macrovascular diseases (T2DM-M). The patients with T2DM-M were assigned to two subgroups based on whether statins were used: group with statins (n=23) and group without statins (n=20).Results Compared with the controls and T2DM, the MFI of CD55 positive neutrophils was significantly lower in T2DM-M (P=0.049 vs controls and P=0.033 vs T2DM); similarly, the MFI of CD59 positive monocytes was also lower in T2DM-M (P=0.038 vs controls and P=0.043 vs T2DM). The MFI of CD59 positive neutrophils in T2DM-M was lower than in T2DM (P=0.032). The levels of CD55 and CD59 were negatively associated with age and blood pressure (r=-0.245--0.352, P=0.041-0.003), but not acute-phase reactants and carotid artery intima-media thickness. The levels of CD55 and CD59 increased after treatment with statins, but the results were not significantly different (P >0.05).Conclusions CD55 and CD59 expressions on peripheral blood leucocytes are decreased in T2DM patients with macrovascular diseases. The results suggest that the decreased levels of complement regulatory proteins might play an important role in diabetic macrovascular

  11. Decreased PARP and procaspase-2 protein levels are associated with cellular drug resistance in childhood acute lymphoblastic leukemia.

    Science.gov (United States)

    Holleman, Amy; den Boer, Monique L; Kazemier, Karin M; Beverloo, H Berna; von Bergh, Anne R M; Janka-Schaub, Gritta E; Pieters, Rob

    2005-09-01

    Drug resistance in childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) is associated with impaired ability to induce apoptosis. To elucidate causes of apoptotic defects, we studied the protein expression of Apaf-1, procaspases-2, -3, -6, -7, -8, -10, and poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) in cells from children with acute lymphoblastic leukemia (ALL; n = 43) and acute myeloid leukemia (AML; n = 10). PARP expression was present in all B-lineage samples, but absent in 4 of 15 T-lineage ALL samples and 3 of 10 AML cases, which was not caused by genomic deletions. PARP expression was a median 7-fold lower in T-lineage ALL (P < .001) and 10-fold lower in AML (P < .001) compared with B-lineage ALL. PARP expression was 4-fold lower in prednisolone, vincristine and L-asparaginase (PVA)-resistant compared with PVA-sensitive ALL patients (P < .001). Procaspase-2 expression was 3-fold lower in T-lineage ALL (P = .022) and AML (P = .014) compared with B-lineage ALL. In addition, procaspase-2 expression was 2-fold lower in PVA-resistant compared to PVA-sensitive ALL patients (P = .042). No relation between apoptotic protease-activating factor 1 (Apaf-1), procaspases-3, -6, -7, -8, -10, and drug resistance was found. In conclusion, low baseline expression of PARP and procaspase-2 is related to cellular drug resistance in childhood acute lymphoblastic leukemia.

  12. Intramolecular and intermolecular fluorescence resonance energy transfer in fluorescent protein-tagged Na-K-Cl cotransporter (NKCC1): sensitivity to regulatory conformational change and cell volume.

    Science.gov (United States)

    Pedersen, Meike; Carmosino, Monica; Forbush, Biff

    2008-02-01

    To examine the structure and function of the Na-K-Cl cotransporter, NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Fluorescent protein tags were added at the N-terminal residue between the regulatory domain and the membrane domain and within a poorly conserved region of the C terminus. Both singly and doubly tagged NKCC1s were appropriately trafficked to the cell membrane and were fully functional; regulation was normal except when YFP was inserted near the regulatory domain, in which case activation occurred only upon incubation with calyculin A. Quenching of YFP fluorescence by Cl(-) provided a ratiometric indicator of intracellular [Cl(-)]. All of the CFP/YFP NKCC pairs exhibited some level of FRET, demonstrating the presence of dimers or higher multimers in functioning NKCC1. With YFP near the regulatory domain and CFP in the C terminus, we recorded a 6% FRET change signaling the regulatory phosphorylation event. On the other hand, when the probe was placed at the extreme N terminus, such changes were not seen, presumably due to the length and predicted flexibility of the N terminus. Substantial FRET changes were observed contemporaneous with cell volume changes, possibly reflective of an increase in molecular crowding upon cell shrinkage.

  13. Role of 72 kDa protein of Leptospira interrogans as a diagnostic marker in acute leptospirosis

    Directory of Open Access Journals (Sweden)

    M Riazi

    2014-01-01

    Full Text Available Background & objectives: Leptospirosis is a widespread zoonotic disease and a public health problem, particularly in tropical and subtropical countries. Varied clinical manifestations of the disease frequently lead to misdiagnosis resulting in life-threatening multi-organ complications. Therefore, early laboratory investigation using an appropriate diagnostic approach is crucial. In the present study, a potential protein marker was identified and evaluated for its usefulness in the serodiagnosis of acute leptospirosis. Methods: Leptospira interrogans serovar Icterohaemorrhagiae (L44, which represents a commonly prevalent serovar in Malaysia, was cultivated for preparation of sequential protein extract (SEQ. SDS-PAGE and immunoblotting were performed with a serum panel comprising confirmed cases of leptospirosis and controls (n=42 each. Identification and characterization of the highest scoring protein from the antigenic band was performed. Subsequently based on the nucleotide coding sequence of the protein, the corresponding recombinant protein was custom-produced. It was then evaluated for sensitivity and specificity by testing against 20 serum samples from leptospirosis patients and 32 from controls. Results: Among the antigenic components, a 72kDa protein band demonstrated significant sensitivity (83.3% and specificity (95.2% for the detection of specific anti-leptospiral IgM antibodies. The protein was identified by mass-spectrometry analysis as heat shock protein DnaK of L. interrogans. Recombinant form of the protein (r72SEQ showed 85 per cent sensitivity and 81 per cent specificity for the detection of specific anti-leptospiral IgM antibodies. Interpretation & conclusions: The findings of our study indicate that a protein (72kDa of L. interrogans has the potential utility of being used for the diagnosis of acute leptospirosis. Further studies need to be done to confirm these findings.

  14. Novel 5' untranslated region directed blockers of iron-regulatory protein-1 dependent amyloid precursor protein translation: implications for down syndrome and Alzheimer's disease.

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    Sanghamitra Bandyopadhyay

    Full Text Available We reported that iron influx drives the translational expression of the neuronal amyloid precursor protein (APP, which has a role in iron efflux. This is via a classic release of repressor interaction of APP mRNA with iron-regulatory protein-1 (IRP1 whereas IRP2 controls the mRNAs encoding the L- and H-subunits of the iron storage protein, ferritin. Here, we identified thirteen potent APP translation blockers that acted selectively towards the uniquely configured iron-responsive element (IRE RNA stem loop in the 5' untranslated region (UTR of APP mRNA. These agents were 10-fold less inhibitory of 5'UTR sequences of the related prion protein (PrP mRNA. Western blotting confirmed that the 'ninth' small molecule in the series selectively reduced neural APP production in SH-SY5Y cells at picomolar concentrations without affecting viability or the expression of α-synuclein and ferritin. APP blocker-9 (JTR-009, a benzimidazole, reduced the production of toxic Aβ in SH-SY5Y neuronal cells to a greater extent than other well tolerated APP 5'UTR-directed translation blockers, including posiphen, that were shown to limit amyloid burden in mouse models of Alzheimer's disease (AD. RNA binding assays demonstrated that JTR-009 operated by preventing IRP1 from binding to the IRE in APP mRNA, while maintaining IRP1 interaction with the H-ferritin IRE RNA stem loop. Thus, JTR-009 constitutively repressed translation driven by APP 5'UTR sequences. Calcein staining showed that JTR-009 did not indirectly change iron uptake in neuronal cells suggesting a direct interaction with the APP 5'UTR. These studies provide key data to develop small molecules that selectively reduce neural APP and Aβ production at 10-fold lower concentrations than related previously characterized translation blockers. Our data evidenced a novel therapeutic strategy of potential impact for people with trisomy of the APP gene on chromosome 21, which is a phenotype long associated with Down

  15. Novel 5' untranslated region directed blockers of iron-regulatory protein-1 dependent amyloid precursor protein translation: implications for down syndrome and Alzheimer's disease.

    Science.gov (United States)

    Bandyopadhyay, Sanghamitra; Cahill, Catherine; Balleidier, Amelie; Huang, Conan; Lahiri, Debomoy K; Huang, Xudong; Rogers, Jack T

    2013-01-01

    We reported that iron influx drives the translational expression of the neuronal amyloid precursor protein (APP), which has a role in iron efflux. This is via a classic release of repressor interaction of APP mRNA with iron-regulatory protein-1 (IRP1) whereas IRP2 controls the mRNAs encoding the L- and H-subunits of the iron storage protein, ferritin. Here, we identified thirteen potent APP translation blockers that acted selectively towards the uniquely configured iron-responsive element (IRE) RNA stem loop in the 5' untranslated region (UTR) of APP mRNA. These agents were 10-fold less inhibitory of 5'UTR sequences of the related prion protein (PrP) mRNA. Western blotting confirmed that the 'ninth' small molecule in the series selectively reduced neural APP production in SH-SY5Y cells at picomolar concentrations without affecting viability or the expression of α-synuclein and ferritin. APP blocker-9 (JTR-009), a benzimidazole, reduced the production of toxic Aβ in SH-SY5Y neuronal cells to a greater extent than other well tolerated APP 5'UTR-directed translation blockers, including posiphen, that were shown to limit amyloid burden in mouse models of Alzheimer's disease (AD). RNA binding assays demonstrated that JTR-009 operated by preventing IRP1 from binding to the IRE in APP mRNA, while maintaining IRP1 interaction with the H-ferritin IRE RNA stem loop. Thus, JTR-009 constitutively repressed translation driven by APP 5'UTR sequences. Calcein staining showed that JTR-009 did not indirectly change iron uptake in neuronal cells suggesting a direct interaction with the APP 5'UTR. These studies provide key data to develop small molecules that selectively reduce neural APP and Aβ production at 10-fold lower concentrations than related previously characterized translation blockers. Our data evidenced a novel therapeutic strategy of potential impact for people with trisomy of the APP gene on chromosome 21, which is a phenotype long associated with Down syndrome (DS

  16. The role and importance of glycosylation of acute phase proteins with focus on alpha-1 antitrypsin in acute and chronic inflammatory conditions.

    Science.gov (United States)

    McCarthy, Cormac; Saldova, Radka; Wormald, Mark R; Rudd, Pauline M; McElvaney, Noel G; Reeves, Emer P

    2014-07-03

    Acute phase proteins (APPs) are a group of circulating plasma proteins which undergo changes quantitatively or qualitatively at the time of inflammation. Many of these APPs are glycosylated, and it has been shown that alterations in glycosylation may occur in inflammatory and malignant conditions. Changes in glycosylation have been studied as potential biomarkers in cancer and also in chronic inflammatory conditions and have been shown to correlate with disease severity in certain conditions. Serine protease inhibitors (serpins), many of which are also APPs, are proteins involved in the control of proteases in numerous pathways. Alpha-1 Antitrypsin (AAT) is the most abundant serpin within the circulation and is an APP which has been shown to increase in response to inflammation. The primary role of AAT is maintaining the protease/antiprotease balance in the lung, but it also possesses important anti-inflammatory and immune-modulating properties. Several glycoforms of AAT exist, and they possess differing properties in regard to plasma half-life and stability. Glycosylation may also be important in determining the immune modulatory properties of AAT. The review will focus on the role and importance of glycosylation in acute phase proteins with particular attention to AAT and its use as a biomarker of disease. The review describes the processes involved in glycosylation, how glycosylation changes in differing disease states, and the alterations that occur to glycans of APPs with disease and inflammation. Finally, the review explores the importance of changes in glycosylation of AAT at times of inflammation and in malignant conditions and how this may impact upon the functions of AAT.

  17. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system.

    Directory of Open Access Journals (Sweden)

    Alessia Galgano

    Full Text Available Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3'-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3'-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs.

  18. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system.

    Science.gov (United States)

    Galgano, Alessia; Forrer, Michael; Jaskiewicz, Lukasz; Kanitz, Alexander; Zavolan, Mihaela; Gerber, André P

    2008-09-08

    Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3'-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3'-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs.

  19. The DtxR protein acting as dual transcriptional regulator directs a global regulatory network involved in iron metabolism of Corynebacterium glutamicum

    Directory of Open Access Journals (Sweden)

    Hüser Andrea T

    2006-02-01

    Full Text Available Abstract Background The knowledge about complete bacterial genome sequences opens the way to reconstruct the qualitative topology and global connectivity of transcriptional regulatory networks. Since iron is essential for a variety of cellular processes but also poses problems in biological systems due to its high toxicity, bacteria have evolved complex transcriptional regulatory networks to achieve an effective iron homeostasis. Here, we apply a combination of transcriptomics, bioinformatics, in vitro assays, and comparative genomics to decipher the regulatory network of the iron-dependent transcriptional regulator DtxR of Corynebacterium glutamicum. Results A deletion of the dtxR gene of C. glutamicum ATCC 13032 led to the mutant strain C. glutamicum IB2103 that was able to grow in minimal medium only under low-iron conditions. By performing genome-wide DNA microarray hybridizations, differentially expressed genes involved in iron metabolism of C. glutamicum were detected in the dtxR mutant. Bioinformatics analysis of the genome sequence identified a common 19-bp motif within the upstream region of 31 genes, whose differential expression in C. glutamicum IB2103 was verified by real-time reverse transcription PCR. Binding of a His-tagged DtxR protein to oligonucleotides containing the 19-bp motifs was demonstrated in vitro by DNA band shift assays. At least 64 genes encoding a variety of physiological functions in iron transport and utilization, in central carbohydrate metabolism and in transcriptional regulation are controlled directly by the DtxR protein. A comparison with the bioinformatically predicted networks of C. efficiens, C. diphtheriae and C. jeikeium identified evolutionary conserved elements of the DtxR network. Conclusion This work adds considerably to our currrent understanding of the transcriptional regulatory network of C. glutamicum genes that are controlled by DtxR. The DtxR protein has a major role in controlling the

  20. ITIH4 (inter-alpha-trypsin inhibitor heavy chain 4) is a new acute-phase protein isolated from cattle during experimental infection

    DEFF Research Database (Denmark)

    Pineiro, M.; Andres, M.; Iturralde, M.;

    2004-01-01

    We have isolated from calf serum a protein with an apparent M, of 120,000. The protein was detected by using antibodies against major acute-phase protein in pigs with acute inflammation. The amino acid sequence of an internal fragment revealed that this protein is the bovine counterpart of ITIH4......, the heavy chain 4 of the inter-alpha-trypsin inhibitor family. The response of this protein in the sera was determined for animals during experimental bacterial and viral infections. In the bacterial model, animals were inoculated with a mixture of Actinomyces pyogenes, Fusobacterium necrophorum....... Because of the significant induction of the protein in the animals in the mastitis and BRSV infection models, we can conclude that ITIH4 is a new positive acute-phase protein in cattle....

  1. 细胞周期调控蛋白与肾脏疾病%Cell cycle- regulatory proteins and kidney disease

    Institute of Scientific and Technical Information of China (English)

    秦福芳; 邵凤民

    2011-01-01

    Cell is alwayse going on cell division, proliferation, hypertrophy, necrosis, no matter what physiological reaction or pathology. And those activities are regulated by Cell cycle - regulatory proteins, the relation and relative progress of Cell cycle - regulatory proteins and kidney disease were reviewed in this paper.%无论是生理情况下或病理情况下,细胞都在进行着分裂、增殖、肥大或凋亡与坏死,而这一系列细胞分裂增殖活动受到细胞周期调控蛋白的调节.本文主要就细胞周期调控蛋白与肾脏疾病之间的关系和相关进展作一综述.

  2. MAP kinases Erk1/2 phosphorylate sterol regulatory element-binding protein (SREBP)-1a at serine 117 in vitro.

    Science.gov (United States)

    Roth, G; Kotzka, J; Kremer, L; Lehr, S; Lohaus, C; Meyer, H E; Krone, W; Müller-Wieland, D

    2000-10-27

    Sterol regulatory element-binding protein (SREBP)-1a is a transcription factor sensing cellular cholesterol levels and integrating gene regulatory signals mediated by MAP kinase cascades. Here we report the identification of serine 117 in SREBP-1a as the major phosphorylation site of the MAP kinases Erk1/2. This site was identified by nanoelectrospray mass spectrometry and peptide sequencing of recombinant fusion proteins phosphorylated by Erk1/2 in vitro. Serine 117 was verified as the major phosphorylation site by in vitro mutagenesis. Mutation of serine 117 to alanine abolished Erk2-mediated phosphorylation in vitro and the MAP kinase-related transcriptional activation of SREBP-1a by insulin and platelet-derived growth factor in vivo. Our data indicate that the MAP kinase-mediated effects on SREBP-1a-regulated target genes are linked to this phosphorylation site.

  3. Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum

    OpenAIRE

    Ana ePalomino; Francisco Javier ePavon; Eduardo eBlanco Calvo; Antonia eSerrano; Sergio eArrabal; Patricia eRivera; Antonio eVargas; Ainhoa eBilbao; Leticia eRubio; Fernando eRodriguez de Fonseca; Juan eSuarez

    2014-01-01

    Growing awareness of cerebellar involvement in addiction is based on the cerebellum’s intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB) and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression {cannabinoid recept...

  4. Effects of retinoic acid and hydrogen peroxide on sterol regulatory element-binding protein-1a activation during adipogenic differentiation of 3T3-L1 cells

    OpenAIRE

    Eldaim, Mabrouk A. Abd; Okamatsu-Ogura, Yuko; Terao, Akira; Kimura, Kazuhiro

    2010-01-01

    Both retinoic acid (RA) and oxidative stress (H2O2) increased transcription and cleavage of membrane-bound sterol regulatory element-binding protein (SREBP)-1, leading to enhanced transcription of fatty acid synthase (FAS) in hepatoma cells. On the other hand, RA and H2O2 decreased and increased lipogenesis in adipocytes, respectively, although roles of SREBP-1 activation in these effects remain to be elucidated. To elucidate its involvement, we examined the activation of SREBP...

  5. Impact of Diet Supplemented by Coconut Milk on Corticosterone and Acute Phase Protein Level under High Stocking Density

    Directory of Open Access Journals (Sweden)

    Majid SHAKERI

    2016-05-01

    Full Text Available The purpose of this study was to investigate effects of coconut milk supplementation on corticosterone and acute phase protein level under high stocking density. A total 300 Cobb 500 male chicks were placed in cages and stocked as 10 birds/cage (normal stocking density and 15 birds/cage (high stocking density. The treatments were as (i control diet and stocked at 10 and 15 birds/cage (ii control diet + 3% coconut milk from 1-42 day and stocked at 10 and 15 birds/cage (iii control diet + 5% coconut milk from 1-42 day and stocked at 10 and 15 birds/cage. On day 42, 20 birds per treatment were slaughtered to collect blood samples. The results showed higher level of corticosterone and acute phase protein level in control diet compare to other supplemented diets with coconut milk. In conclusion, coconut milk decreased the level of corticosterone and acute phase protein when chicks were subjected to high stocking density.

  6. Acute post-exercise myofibrillar protein synthesis is not correlated with resistance training-induced muscle hypertrophy in young men.

    Science.gov (United States)

    Mitchell, Cameron J; Churchward-Venne, Tyler A; Parise, Gianni; Bellamy, Leeann; Baker, Steven K; Smith, Kenneth; Atherton, Philip J; Phillips, Stuart M

    2014-01-01

    Muscle hypertrophy following resistance training (RT) involves activation of myofibrillar protein synthesis (MPS) to expand the myofibrillar protein pool. The degree of hypertrophy following RT is, however, highly variable and thus we sought to determine the relationship between the acute activation of MPS and RT-induced hypertrophy. We measured MPS and signalling protein activation after the first session of resistance exercise (RE) in untrained men (n = 23) and then examined the relation between MPS with magnetic resonance image determined hypertrophy. To measure MPS, young men (24±1 yr; body mass index  = 26.4±0.9 kg•m²) underwent a primed constant infusion of L-[ring-¹³C₆] phenylalanine to measure MPS at rest, and acutely following their first bout of RE prior to 16 wk of RT. Rates of MPS were increased 235±38% (PHypertrophy after chronic RT was correlated (r = 0.42, P = 0.05) with phosphorylation of 4E-BP1(Thr37/46) at 1 hour post RE. We conclude that acute measures of MPS following an initial exposure to RE in novices are not correlated with muscle hypertrophy following chronic RT.

  7. The value of pancreatic stone protein in predicting acute appendicitis in patients presenting at the emergency department with abdominal pain

    Directory of Open Access Journals (Sweden)

    Tschuor Christoph

    2012-10-01

    Full Text Available Abstract Background Pancreatic Stone Protein (PSP is a protein naturally produced mainly in the pancreas and the gut. There is evidence from experimental and clinical trials that blood PSP levels rise in the presence of inflammation or infection. However, it is not known whether PSP is superior to other established blood tests (e.g. White Blood Count, Neutrophils or C - reactive protein in predicting appendicitis in patients presenting with abdominal pain and a clinical suspicion of appendicitis at the emergency room. Methods/design The PSP Appendix Trial is a prospective, multi-center, cohort study to assess the value of PSP in the diagnostic workup of acute appendicitis. 245 patients will be prospectively recruited. Interim analysis will be performed once 123 patients are recruited. The primary endpoint of the study concerns the diagnostic accuracy of PSP in predicting acute appendicitis and therefore the evidence of appendicitis on the histopathological specimen after appendectomy. Discussion The PSP Appendix Trial is a prospective, multi-center, cohort study to assess the value of PSP in the diagnostic workup of acute appendicitis. Trial registration ClinicalTrials.gov: NCT01610193; Institution Ethical Board Approval ID: KEKZH- Nr. 2011–0501

  8. Comprehensive behavioral analysis of voltage-gated calcium channel beta-anchoring and -regulatory protein knockout mice

    Directory of Open Access Journals (Sweden)

    Akito eNakao

    2015-06-01

    Full Text Available Calcium (Ca2+ influx through voltage-gated Ca2+ channels (VGCCs induces numerous intracellular events such as neuronal excitability, neurotransmitter release, synaptic plasticity, and gene regulation. It has been shown that genes related to Ca2+ signaling, such as the CACNA1C, CACNB2, and CACNA1I genes that encode VGCC subunits, are associated with schizophrenia and other psychiatric disorders. Recently, VGCC beta-anchoring and -regulatory protein (BARP was identified as a novel regulator of VGCC activity via the interaction of VGCC β subunits. To examine the role of the BARP in higher brain functions, we generated BARP knockout (KO mice and conducted a comprehensive battery of behavioral tests. BARP KO mice exhibited greatly reduced locomotor activity, as evidenced by decreased vertical activity, stereotypic counts in the open field test, and activity level in the home cage, and longer latency to complete a session in spontaneous T-maze alteration test, which reached study-wide significance. Acoustic startle response was also reduced in the mutants. Interestingly, they showed multiple behavioral phenotypes that are seemingly opposite to those seen in the mouse models of schizophrenia and its related disorders, including increased working memory, flexibility, prepulse inhibition, and social interaction, and decreased locomotor activity, though many of these phenotypes are statistically weak and require further replications. These results demonstrate that BARP is involved in the regulation of locomotor activity and, possibly, emotionality. The possibility was also suggested that BARP KO mice may serve as a unique tool for investigating the pathogenesis/pathophysiology of schizophrenia and related disorders. Further evaluation of the molecular and physiological phenotypes of the mutant mice would provide new insights into the role of BARP in higher brain functions.

  9. Sterol regulatory element-binding proteins are regulators of the rat thyroid peroxidase gene in thyroid cells.

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    Christine Rauer

    Full Text Available Sterol regulatory element-binding proteins (SREBPs-1c and -2, which were initially discovered as master transcriptional regulators of lipid biosynthesis and uptake, were recently identified as novel transcriptional regulators of the sodium-iodide symporter gene in the thyroid, which is essential for thyroid hormone synthesis. Based on this observation that SREBPs play a role for thyroid hormone synthesis, we hypothesized that another gene involved in thyroid hormone synthesis, the thyroid peroxidase (TPO gene, is also a target of SREBP-1c and -2. Thyroid epithelial cells treated with 25-hydroxycholesterol, which is known to inhibit SREBP activation, had about 50% decreased mRNA levels of TPO. Similarly, the mRNA level of TPO was reduced by about 50% in response to siRNA mediated knockdown of both, SREBP-1 and SREBP-2. Reporter gene assays revealed that overexpression of active SREBP-1c and -2 causes a strong transcriptional activation of the rat TPO gene, which was localized to an approximately 80 bp region in the intron 1 of the rat TPO gene. In vitro- and in vivo-binding of both, SREBP-1c and SREBP-2, to this region in the rat TPO gene could be demonstrated using gel-shift assays and chromatin immunoprecipitation. Mutation analysis of the 80 bp region of rat TPO intron 1 revealed two isolated and two overlapping SREBP-binding elements from which one, the overlapping SRE+609/InvSRE+614, was shown to be functional in reporter gene assays. In connection with recent findings that the rat NIS gene is also a SREBP target gene in the thyroid, the present findings suggest that SREBPs may be possible novel targets for pharmacological modulation of thyroid hormone synthesis.

  10. Dynamic changes in binding of immunoglobulin heavy chain 3' regulatory region to protein factors during class switching.

    Science.gov (United States)

    Chatterjee, Sanjukta; Ju, Zhongliang; Hassan, Rabih; Volpi, Sabrina A; Emelyanov, Alexander V; Birshtein, Barbara K

    2011-08-19

    The 3' regulatory region (3' RR) of the Igh locus works at long distances on variable region (V(H)) and switch region (I) region promoters to initiate germ line (non-coding) transcription (GT) and promote class switch recombination (CSR). The 3' RR contains multiple elements, including enhancers (hs3a, hs1.2, hs3b, and hs4) and a proposed insulator region containing CTCF (CCCTC-binding factor) binding sites, i.e. hs5/6/7 and the downstream region ("38"). Notably, deletion of each individual enhancer (hs3a-hs4) has no significant phenotypic consequence, suggesting that the 3' RR has considerable structural flexibility in its function. To better understand how the 3' RR functions, we identified transcription factor binding sites and used chromatin immunoprecipitation (ChIP) assays to monitor their occupancy in splenic B cells that initiate GT and undergo CSR (LPS±IL4), are deficient in GT and CSR (p50(-/-)), or do not undergo CSR despite efficient GT (anti-IgM+IL4). Like 3' RR enhancers, hs5-7 and the 38 region were observed to contain multiple Pax5 binding sites (in addition to multiple CTCF sites). We found that the Pax5 binding profile to the 3' RR dynamically changed during CSR independent of the specific isotype to which switching was induced, and binding focused on hs1.2, hs4, and hs7. CTCF-associated and CTCF-independent cohesin interactions were also identified. Our observations are consistent with a scaffold model in which a platform of active protein complexes capable of facilitating GT and CSR can be formed by varying constellations of 3' RR elements.

  11. High concentrations of protein test substances may have non-toxic effects on Daphnia magna: Implications for regulatory study designs and ecological risk assessments for GM crops

    Science.gov (United States)

    Raybould, Alan; Burns, Andrea; Hamer, Mick

    2014-01-01

    Laboratory testing for possible adverse effects of insecticidal proteins on non-target organisms (NTOs) is an important part of many ecological risk assessments for regulatory decision-making about the cultivation of insect-resistant genetically modified (IRGM) crops. To increase confidence in the risk assessments, regulatory guidelines for effects testing specify that representative surrogate species for NTOs are exposed to concentrations of insecticidal proteins that are in excess of worst-case predicted exposures in the field. High concentrations in effects tests are achieved by using protein test substances produced in microbes, such as Escherichia coli. In a study that exposed Daphnia magna to a single high concentration of a microbial test substance containing Vip3Aa20, the insecticidal protein in MIR162 maize, small reductions in growth were observed. These effects were surprising as many other studies strongly suggest that the activity of Vip3Aa20 is limited to Lepidoptera. A plausible explanation for the effect on growth is that high concentrations of test substance have a non-toxic effect on Daphnia, perhaps by reducing its feeding rate. A follow-up study tested that hypothesis by exposing D. magna to several concentrations of Vip3Aa20, and a high concentration of a non-toxic protein, bovine serum albumin (BSA). Vip3Aa20 and BSA had sporadic effects on the reproduction and growth of D. magna. The pattern of the effects suggests that they result from non-toxic effects of high concentrations of protein, and not from toxicity. The implications of these results for regulatory NTO effects testing and ERA of IRGM crops are discussed. PMID:25523175

  12. High concentrations of protein test substances may have non-toxic effects on Daphnia magna: implications for regulatory study designs and ecological risk assessments for GM crops.

    Science.gov (United States)

    Raybould, Alan; Burns, Andrea; Hamer, Mick

    2014-01-01

    Laboratory testing for possible adverse effects of insecticidal proteins on non-target organisms (NTOs) is an important part of many ecological risk assessments for regulatory decision-making about the cultivation of insect-resistant genetically modified (IRGM) crops. To increase confidence in the risk assessments, regulatory guidelines for effects testing specify that representative surrogate species for NTOs are exposed to concentrations of insecticidal proteins that are in excess of worst-case predicted exposures in the field. High concentrations in effects tests are achieved by using protein test substances produced in microbes, such as Escherichia coli. In a study that exposed Daphnia magna to a single high concentration of a microbial test substance containing Vip3Aa20, the insecticidal protein in MIR162 maize, small reductions in growth were observed. These effects were surprising as many other studies strongly suggest that the activity of Vip3Aa20 is limited to Lepidoptera. A plausible explanation for the effect on growth is that high concentrations of test substance have a non-toxic effect on Daphnia, perhaps by reducing its feeding rate. A follow-up study tested that hypothesis by exposing D. magna to several concentrations of Vip3Aa20, and a high concentration of a non-toxic protein, bovine serum albumin (BSA). Vip3Aa20 and BSA had sporadic effects on the reproduction and growth of D. magna. The pattern of the effects suggests that they result from non-toxic effects of high concentrations of protein, and not from toxicity. The implications of these results for regulatory NTO effects testing and ERA of IRGM crops are discussed.

  13. Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

    Energy Technology Data Exchange (ETDEWEB)

    Colson, Brett A.; Locher, Matthew R.; Bekyarova, Tanya; Patel, Jitandrakumar R.; Fitzsimons, Daniel P.; Irving, Thomas C.; Moss, Richard L. (IIT); (UW-MED)

    2010-05-25

    Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca{sup 2+} sensitivity of force (pCa{sub 50}), PKA treatment has been shown to decrease pCa{sub 50}, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca{sup 2+}-independent force and maximum Ca{sup 2+}-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I{sub 1,1}/I{sub 1,0}) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d{sub 1,0}) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I{sub 1,1}/I{sub 1,0} and, as hypothesized, treatment with MLCK also increased I{sub 1,1}/I{sub 1,0}, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by {approx}2 nm ({Delta} 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca{sup 2+} sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

  14. Identification of a phosphorylation-dependent nuclear localization motif in interferon regulatory factor 2 binding protein 2.

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    Allen C T Teng

    Full Text Available BACKGROUND: Interferon regulatory factor 2 binding protein 2 (IRF2BP2 is a muscle-enriched transcription factor required to activate vascular endothelial growth factor-A (VEGFA expression in muscle. IRF2BP2 is found in the nucleus of cardiac and skeletal muscle cells. During the process of skeletal muscle differentiation, some IRF2BP2 becomes relocated to the cytoplasm, although the functional significance of this relocation and the mechanisms that control nucleocytoplasmic localization of IRF2BP2 are not yet known. METHODOLOGY/PRINCIPAL FINDINGS: Here, by fusing IRF2BP2 to green fluorescent protein and testing a series of deletion and site-directed mutagenesis constructs, we mapped the nuclear localization signal (NLS to an evolutionarily conserved sequence (354ARKRKPSP(361 in IRF2BP2. This sequence corresponds to a classical nuclear localization motif bearing positively charged arginine and lysine residues. Substitution of arginine and lysine with negatively charged aspartic acid residues blocked nuclear localization. However, these residues were not sufficient because nuclear targeting of IRF2BP2 also required phosphorylation of serine 360 (S360. Many large-scale phosphopeptide proteomic studies had reported previously that serine 360 of IRF2BP2 is phosphorylated in numerous human cell types. Alanine substitution at this site abolished IRF2BP2 nuclear localization in C(2C(12 myoblasts and CV1 cells. In contrast, substituting serine 360 with aspartic acid forced nuclear retention and prevented cytoplasmic redistribution in differentiated C(2C(12 muscle cells. As for the effects of these mutations on VEGFA promoter activity, the S360A mutation interfered with VEGFA activation, as expected. Surprisingly, the S360D mutation also interfered with VEGFA activation, suggesting that this mutation, while enforcing nuclear entry, may disrupt an essential activation function of IRF2BP2. CONCLUSIONS/SIGNIFICANCE: Nuclear localization of IRF2BP2 depends on

  15. Regulatory circuit for responses of nitrogen catabolic gene expression to the GLN3 and DAL80 proteins and nitrogen catabolite repression in Saccharomyces cerevisiae.

    OpenAIRE

    Daugherty, J R; Rai, R; el Berry, H M; Cooper, T. G.

    1993-01-01

    We demonstrate that expression of the UGA1, CAN1, GAP1, PUT1, PUT2, PUT4, and DAL4 genes is sensitive to nitrogen catabolite repression. The expression of all these genes, with the exception of UGA1 and PUT2, also required a functional GLN3 protein. In addition, GLN3 protein was required for expression of the DAL1, DAL2, DAL7, GDH1, and GDH2 genes. The UGA1, CAN1, GAP1, and DAL4 genes markedly increased their expression when the DAL80 locus, encoding a negative regulatory element, was disrupt...

  16. RNAi mediated acute depletion of Retinoblastoma protein (pRb promotes aneuploidy in human primary cells via micronuclei formation

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    Iovino Flora

    2009-11-01

    Full Text Available Abstract Background Changes in chromosome number or structure as well as supernumerary centrosomes and multipolar mitoses are commonly observed in human tumors. Thus, centrosome amplification and mitotic checkpoint dysfunctions are believed possible causes of chromosomal instability. The Retinoblastoma tumor suppressor (RB participates in the regulation of synchrony between DNA synthesis and centrosome duplication and it is involved in transcription regulation of some mitotic genes. Primary human fibroblasts were transfected transiently with short interfering RNA (siRNA specific for human pRb to investigate the effects of pRb acute loss on chromosomal stability. Results Acutely pRb-depleted fibroblasts showed altered expression of genes necessary for cell cycle progression, centrosome homeostasis, kinetochore and mitotic checkpoint proteins. Despite altered expression of genes involved in the Spindle Assembly Checkpoint (SAC the checkpoint seemed to function properly in pRb-depleted fibroblasts. In particular AURORA-A and PLK1 overexpression suggested that these two genes might have a role in the observed genomic instability. However, when they were post-transcriptionally silenced in pRb-depleted fibroblasts we did not observe reduction in the number of aneuploid cells. This finding suggests that overexpression of these two genes did not contribute to genomic instability triggered by RB acute loss although it affected cell proliferation. Acutely pRb-depleted human fibroblasts showed the presence of micronuclei containing whole chromosomes besides the presence of supernumerary centrosomes and aneuploidy. Conclusion Here we show for the first time that RB acute loss triggers centrosome amplification and aneuploidy in human primary fibroblasts. Altogether, our results suggest that pRb-depleted primary human fibroblasts possess an intact spindle checkpoint and that micronuclei, likely caused by mis-attached kinetochores that in turn trigger

  17. Does C-reactive Protein Add Prognostic Value to GRACE Score in Acute Coronary Syndromes?

    Energy Technology Data Exchange (ETDEWEB)

    Correia, Luis Cláudio Lemos, E-mail: lccorreia@terra.com.br; Vasconcelos, Isis; Garcia, Guilherme; Kalil, Felipe; Ferreira, Felipe; Silva, André; Oliveira, Ruan; Carvalhal, Manuela; Freitas, Caio; Noya-Rabelo, Márcia Maria [Escola Bahiana de Medicina e Saúde Pública, Salvador, BA (Brazil); Hospital São Rafael, Salvador, BA (Brazil)

    2014-05-15

    The incremental prognostic value of plasma levels of C-reactive protein (CRP) in relation to GRACE score has not been established in patients with acute coronary syndrome (ACS) with non-ST segment elevation. To test the hypothesis that CRP measurements at admission increases the prognostic value of GRACE score in patients with ACS. A total of 290 subjects, consecutively admitted for ACS, with plasma material obtained upon admission CRP measurement using a high-sensitivity method (nephelometry) were studied. Cardiovascular outcomes during hospitalization were defined by the combination of death, nonfatal myocardial infarction or nonfatal refractory angina. The incidence of cardiovascular events during hospitalization was 15% (18 deaths, 11 myocardial infarctions, 13 angina episodes) with CRP showing C-statistics of 0.60 (95% CI = 0.51-0.70, p = 0.034) in predicting these outcomes. After adjustment for the GRACE score, elevated CRP (defined as the best cutoff point) tended to be associated with hospital events (OR = 1.89, 95% CI = 0.92 to 3.88, p = 0.08). However, the addition of the variable elevated CRP in the GRACE model did not result in significant increase in C-statistics, which ranged from 0.705 to 0.718 (p = 0.46). Similarly, there was no significant reclassification of risk with the addition of CRP in the predictor model (net reclassification = 5.7 %, p = 0.15). Although CRP is associated with hospital outcomes, this inflammatory marker does not increase the prognostic value of the GRACE score.

  18. Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia

    Energy Technology Data Exchange (ETDEWEB)

    Benny Klimek, Margaret E.; Aydogdu, Tufan [Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL (United States); Link, Majik J.; Pons, Marianne [Molecular Oncology Program, Division of Surgical Oncology, DeWitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, Miami, FL (United States); Koniaris, Leonidas G. [Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL (United States); Molecular Oncology Program, Division of Surgical Oncology, DeWitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, Miami, FL (United States); Molecular Oncology and Experimental Therapeutics Program, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL (United States); Zimmers, Teresa A., E-mail: tzimmers@med.miami.edu [Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL (United States); Molecular Oncology Program, Division of Surgical Oncology, DeWitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, Miami, FL (United States); Molecular Oncology and Experimental Therapeutics Program, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL (United States)

    2010-01-15

    Cachexia, progressive loss of fat and muscle mass despite adequate nutrition, is a devastating complication of cancer associated with poor quality of life and increased mortality. Myostatin is a potent tonic muscle growth inhibitor. We tested how myostatin inhibition might influence cancer cachexia using genetic and pharmacological approaches. First, hypermuscular myostatin null mice were injected with Lewis lung carcinoma or B16F10 melanoma cells. Myostatin null mice were more sensitive to tumor-induced cachexia, losing more absolute mass and proportionately more muscle mass than wild-type mice. Because myostatin null mice lack expression from development, however, we also sought to manipulate myostatin acutely. The histone deacetylase inhibitor Trichostatin A has been shown to increase muscle mass in normal and dystrophic mice by inducing the myostatin inhibitor, follistatin. Although Trichostatin A administration induced muscle growth in normal mice, it failed to preserve muscle in colon-26 cancer cachexia. Finally we sought to inhibit myostatin and related ligands by administration of the Activin receptor extracellular domain/Fc fusion protein, ACVR2B-Fc. Systemic administration of ACVR2B-Fc potently inhibited muscle wasting and protected adipose stores in both colon-26 and Lewis lung carcinoma cachexia, without affecting tumor growth. Enhanced cachexia in myostatin knockouts indicates that host-derived myostatin is not the sole mediator of muscle wasting in cancer. More importantly, skeletal muscle preservation with ACVR2B-Fc establishes that targeting myostatin-family ligands using ACVR2B-Fc or related molecules is an important and potent therapeutic avenue in cancer cachexia.

  19. Dexamethasone mediates protection against acute pancreatitis via upregulation of pancreatitis-associated proteins

    Institute of Scientific and Technical Information of China (English)

    Emad Kandil; Yin-Yao Lin; Martin H Bluth; Hong Zhang; Gabriel Levi; Michael E Zenilman

    2006-01-01

    AIM:To examine the influence of dexamethasone on pancreatitis-associated protein (PAP) gene expression using both in vitro and in vivo models of acute pancreatitis and to study how PAP gene expression correlates with severity of pancreatitis.METHODS:In vifro, IL-6 stimulated pancreas acinar AR42J cells were cultured with increasing concentrations of dexamethasone and assayed for PAP expression (RT-PCR). In vivo, pancreatitis was induced in rats by retrograde injection of 40 g/L taurocholate into the pancreatic duct. Animals were pretreated with dexamethasone (2 mg/kg) daily or saline for 4 d.Pancreata and serum were harvested after 24 h and gene expression levels of PAP Ⅰ , Ⅱ and Ⅲ were measured by RT-PCR. Severity of pancreatitis was based on serum amylase, pancreatic wet weight, and histopathological score.RESULTS:In vitro, dexamethasone and IL-6 induced a marked transcription of PAP Ⅰ, Ⅱ and Ⅲ genes in AR42J cells at 24 h (P < 0.05 for all comparisons). In vivo,pancreas mRNA levels of PAP Ⅰ, Ⅱ or Ⅲ increased by 2.6-fold, 1.9-fold, and 1.3-fold respectively after dexamethasone treatment, compared with saline treated animals. Serum amylase levels and edema were significantly lower in the dexamethasone group compared with the saline group. Histopathologic evaluation revealed less inflammation and necrosis in pancreata obtained from dexamethasone treated animals (P < 0.05).CONCLUSION:Dexamethasone significantly decreases the severity of pancreatitis. The protective mechanism of dexamethasone may be via upregulating PAP gene expression during injury.

  20. α -Actinin TvACTN3 of Trichomonas vaginalis is an RNA-binding protein that could participate in its posttranscriptional iron regulatory mechanism.

    Science.gov (United States)

    Calla-Choque, Jaeson Santos; Figueroa-Angulo, Elisa Elvira; Ávila-González, Leticia; Arroyo, Rossana

    2014-01-01

    Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system.

  1. Yeast sterol regulatory element-binding protein (SREBP) cleavage requires Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing subunit of the Golgi Dsc E3 ligase.

    Science.gov (United States)

    Stewart, Emerson V; Lloyd, S Julie-Ann; Burg, John S; Nwosu, Christine C; Lintner, Robert E; Daza, Riza; Russ, Carsten; Ponchner, Karen; Nusbaum, Chad; Espenshade, Peter J

    2012-01-01

    Schizosaccharomyces pombe Sre1 is a membrane-bound transcription factor that controls adaptation to hypoxia. Like its mammalian homolog, sterol regulatory element-binding protein (SREBP), Sre1 activation requires release from the membrane. However, in fission yeast, this release occurs through a strikingly different mechanism that requires the Golgi Dsc E3 ubiquitin ligase complex and the proteasome. The mechanistic details of Sre1 cleavage, including the link between the Dsc E3 ligase complex and proteasome, are not well understood. Here, we present results of a genetic selection designed to identify additional components required for Sre1 cleavage. From the selection, we identified two new components of the fission yeast SREBP pathway: Dsc5 and Cdc48. The AAA (ATPase associated with diverse cellular activities) ATPase Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing protein, interact with known Dsc complex components and are required for SREBP cleavage. These findings provide a mechanistic link between the Dsc E3 ligase complex and the proteasome in SREBP cleavage and add to a growing list of similarities between the Dsc E3 ligase and membrane E3 ligases involved in endoplasmic reticulum-associated degradation.

  2. Acute post-exercise myofibrillar protein synthesis is not correlated with resistance training-induced muscle hypertrophy in young men.

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    Cameron J Mitchell

    Full Text Available Muscle hypertrophy following resistance training (RT involves activation of myofibrillar protein synthesis (MPS to expand the myofibrillar protein pool. The degree of hypertrophy following RT is, however, highly variable and thus we sought to determine the relationship between the acute activation of MPS and RT-induced hypertrophy. We measured MPS and signalling protein activation after the first session of resistance exercise (RE in untrained men (n = 23 and then examined the relation between MPS with magnetic resonance image determined hypertrophy. To measure MPS, young men (24±1 yr; body mass index  = 26.4±0.9 kg•m² underwent a primed constant infusion of L-[ring-¹³C₆] phenylalanine to measure MPS at rest, and acutely following their first bout of RE prior to 16 wk of RT. Rates of MPS were increased 235±38% (P<0.001 above rest 60-180 min post-exercise and 184±28% (P = 0.037 180-360 min post exercise. Quadriceps volume increased 7.9±1.6% (-1.9-24.7% (P<0.001 after training. There was no correlation between changes in quadriceps muscle volume and acute rates of MPS measured over 1-3 h (r = 0.02, 3-6 h (r = 0.16 or the aggregate 1-6 h post-exercise period (r = 0.10. Hypertrophy after chronic RT was correlated (r = 0.42, P = 0.05 with phosphorylation of 4E-BP1(Thr37/46 at 1 hour post RE. We conclude that acute measures of MPS following an initial exposure to RE in novices are not correlated with muscle hypertrophy following chronic RT.

  3. Inducing Acute Traumatic Coagulopathy In Vitro: The Effects of Activated Protein C on Healthy Human Whole Blood.

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    Benjamin M Howard

    Full Text Available Acute traumatic coagulopathy has been associated with shock and tissue injury, and may be mediated via activation of the protein C pathway. Patients with acute traumatic coagulopathy have prolonged PT and PTT, and decreased activity of factors V and VIII; they are also hypocoagulable by thromboelastometry (ROTEM and other viscoelastic assays. To test the etiology of this phenomenon, we hypothesized that such coagulopathy could be induced in vitro in healthy human blood with the addition of activated protein C (aPC.Whole blood was collected from 20 healthy human subjects, and was "spiked" with increasing concentrations of purified human aPC (control, 75, 300, 2000 ng/mL. PT/PTT, factor activity assays, and ROTEM were performed on each sample. Mixed effect regression modeling was performed to assess the association of aPC concentration with PT/PTT, factor activity, and ROTEM parameters.In all subjects, increasing concentrations of aPC produced ROTEM tracings consistent with traumatic coagulopathy. ROTEM EXTEM parameters differed significantly by aPC concentration, with stepwise prolongation of clotting time (CT and clot formation time (CFT, decreased alpha angle (α, impaired early clot formation (a10 and a20, and reduced maximum clot firmness (MCF. PT and PTT were significantly prolonged at higher aPC concentrations, with corresponding significant decreases in factor V and VIII activity.A phenotype of acute traumatic coagulopathy can be induced in healthy blood by the in vitro addition of aPC alone, as evidenced by viscoelastic measures and confirmed by conventional coagulation assays and factor activity. This may lend further mechanistic insight to the etiology of coagulation abnormalities in trauma, supporting the central role of the protein C pathway. Our findings also represent a model for future investigations in the diagnosis and treatment of acute traumatic coagulopathy.

  4. Disaturated-phosphatidylcholine and Surfactant protein-B turnover in human acute lung injury and in control patients

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    Rizzi Sabina

    2011-03-01

    Full Text Available Abstract Background Patients with Adult Respiratory Distress Syndrome (ARDS and Acute Lung Injury (ALI have low concentrations of disaturated-phosphatidylcholine and surfactant protein-B in bronchoalveolar lavage fluid. No information is available on their turnover. Objectives To analyze disaturated-phosphatidylcholine and surfactant protein-B turnover in patients with ARDS/ALI and in human adults with normal lungs (controls. Methods 2H2O as precursor of disaturated-phosphatidylcholine-palmitate and 113C-Leucine as precursor of surfactant protein-B were administered intravenously to 12 patients with ARDS/ALI and to 8 controls. Disaturated-phosphatidylcholine and surfactant protein-B were isolated from serial tracheal aspirates, and their fractional synthetic rate was derived from the 2H and 13C enrichment curves, obtained by gas chromatography mass spectrometry. Disaturated-phosphatidylcholine, surfactant protein-B, and protein concentrations in tracheal aspirates were also measured. Results 1 Surfactant protein-B turned over at faster rate than disaturated-phosphatidylcholine both in ARDS/ALI patients and in controls. 2 In patients with ARDS/ALI the fractional synthesis rate of disaturated-phosphatidylcholine was 3.1 times higher than in controls (p Conclusions 1 Disaturated-phosphatidylcholine and surfactant protein-B have a different turnover both in healthy and diseased lungs. 2 In ARDS/ALI the synthesis of these two surfactant components may be differently regulated.

  5. Changes of inducible protein-10 and regulated upon activation, normal T cell expressed and secreted protein in acute rejection of pancreas transplantation in rats

    Institute of Scientific and Technical Information of China (English)

    Jun Zhu; Ze-Kuan Xu; Yi Miao; Xun-Liang Liu; Hong Zhang

    2006-01-01

    AIM: To investigate the role of IFN-γ inducible protein -10 (Ip-10) and regulated upon activation, normal T cell expressed and secreted (RANTES) protein in acute pancreatic allograft rejection in rats.METHODS: An experimental pancreas transplantation model was established using diabetic SD rats as the recipient, induced by applying streptozocin (STZ).Pancreas transplantation was performed with a physiologic method of portal venous and enteric drainage. Rats were divided into two groups, isograft group (group A, n = 24) and allograft group (group B, n = 24) in which either healthy SD rats or Wistar rats served as donors, respectively. Twelve diabetic or healthy SD rats were used as controls. At d 1, 4, 7, and 10 post transplantation, serum IP-10 and RANTES were assessed by ELISA and their expression in the allografts was determined by immunohistochemistry.RESULTS: In group B (allograft group), the development of acute rejection was significantly correlated with increased serum concentration and tissue expression of IP-10 and RANTES, with a peak level at d 7 post transplantation. In contrast, there was no obvious change before and after transplantation in group A (isograft group).CONCLUSION: Our study suggests a possible role of IP-10 and RANTES in acute rejection and early monitoring of chemokines may be helpful in predicting the outcome of pancreas transplantation.

  6. Requirement for the POZ/BTB protein NAC1 in acute but not chronic psychomotor stimulant response.

    Science.gov (United States)

    Mackler, Scott; Pacchioni, Alejandra; Degnan, Ryan; Homan, Ying; Conti, Alana C; Kalivas, Peter; Blendy, Julie A

    2008-02-11

    NAC1 is a novel member of the POZ/BTB (Pox virus and Zinc finger/Bric-a-bracTramtrack Broad complex) but varies from other proteins of this class in that it lacks the characteristic DNA-binding motif, suggesting a novel role. We have employed constitutive gene deletion to elucidate the role of NAC1 in vivo. Nac1 mutant mice are viable with no obvious developmental or physiological impairments. Previous studies suggest a role for NAC1 in cocaine-mediated behaviors. Therefore, we evaluated a variety of behaviors associated with psychomotor stimulant effects in Nac1 mutant mice. Acute locomotor activating effects of cocaine or amphetamine are absent in Nac1 mutant mice, however longer exposure to these psychomotor stimulants result in the development of behavioral sensitization. Acute rewarding properties of cocaine and amphetamine are also blunted in mutant mice, yet repeated exposure resulted in conditioned place preference similar to that observed in wild-type mice. Lastly, increases in extracellular dopamine in the nucleus accumbens, which accompany acute cocaine administration, are blunted in mutant mice, but following chronic cocaine extracellular dopamine levels are increased to the same extent as in wild-type mice. Together these data indicate involvement of NAC1 in the acute behavioral and neurochemical responses to psychomotor stimulants.

  7. Regulatory agencies and regulatory risk

    OpenAIRE

    Knieps, Günter; Weiß, Hans-Jörg

    2007-01-01

    The aim of this paper is to show that regulatory risk is due to the discretionary behaviour of regulatory agencies, caused by a too extensive regulatory mandate provided by the legislator. The normative point of reference and a behavioural model of regulatory agencies based on the positive theory of regulation are presented. Regulatory risk with regard to the future behaviour of regulatory agencies is modelled as the consequence of the ex ante uncertainty about the relative influence of inter...

  8. The number of regulatory T cells in transbronchial lung allograft biopsies is related to FoxP3 mRNA levels in bronchoalveolar lavage fluid and to the degree of acute cellular rejection

    DEFF Research Database (Denmark)

    Krustrup, Dorrit; Madsen, Caroline B; Iversen, Martin;

    2013-01-01

    The transcription factor Forkhead Box P3 (FoxP3) is a marker of regulatory T cells (Tregs) - a subset of T cells known to suppress a wide range of immune responses. These cells are considered to be pivotal for the induction of tolerance to donor antigens in human allografts. We aimed to correlate...... the number of lymphocytes expressing FoxP3 in transbronchial biopsies from lung allografts with the FoxP3 expression in bronchoalveolar lavage fluid (BALF). In addition, we aimed to correlate the number of FoxP3+ cells in transbronchial biopsies with the degree of acute cellular rejection in lung allografts....

  9. Changes of serum high sensitive C-reactive protein in patients with acute cerebral infarction

    Institute of Scientific and Technical Information of China (English)

    Yan Du; Yan Ren; Ying Li

    2007-01-01

    BACKGROUND: Serum high sensitive C-reactive protein (hs-CRP), which regards as a high sensitive mark of systemic inflammatory response syndrome, can provide a lot of valuable information for the treatment and prognosis of cerebrovascular disease.OBJECTIVE: To observe the differences of blood glucose, lipid, homocysteine and previous disease history among patients with acute cerebral infarction at various levels of hs-CRP and compare changes of hs-CRP of patients with various degrees ofneurologic impairment.DESIGN: Contrast observation.SETTING: Department of Neurology, Shenzhou Hospital, Shenyang Medical College.PARTICIPANTS: A total of 102 patients with acute cerebral infarction were selected from Department of Neurology, Shenzhou Hospital of Shenyang Medical College from February 2005 to September 2006,including 55 males and 47 females aged from 55 to 86 years. All accepted patients met the diagnostic criteria of cerebral infarction established by the Fourth National Cerebrovascular Disease Academic Meeting and were diagnosed with CT or MRI examination. All patients provided the confirmed consent. Based on clinical criteria of neurologic impairment established by the Fourth National Cerebrovascular Disease Academic Meeting, patients were randomly divided into mild group (0- 15 points, n =46), moderate group (16- 30points, n =38) and severe group (31 - 45 points, n =18). In addition, based on hs-CRP level within 72 hours,patients were divided into normal group (hs-CRP ≤ 3 mg/L, n =53) and increasing group (hs-CRP > 3 mg/L,n =49).METHODS: ① 2 mL venous blood was selected from hospitalized patients in the next morning to separate serum. Quantitative measurement of hs-CRP was dealt with Latex Enhnced Turbidimetric Immunoassay (LETIA). ② Fasting venous blood was colleted from hospitalized patients in the next morning to measure numeration of white blood cells, fibrinogen, blood glucose, total cholesterol (TC), triacylglycerol (TG), high density lipoprotein

  10. Gp130-dependent release of acute phase proteins is linked to the activation of innate immune signaling pathways.

    Directory of Open Access Journals (Sweden)

    Maren Luchtefeld

    Full Text Available BACKGROUND: Elevated levels of acute phase proteins (APP are often found in patients with cardiovascular diseases. In a previous study, we demonstrated the importance of the IL-6-gp130 axis -as a key regulator of inflammatory acute phase signaling in hepatocytes-for the development of atherosclerosis. BACKGROUND/PRINCIPAL FINDINGS: Gp130-dependent gene expression was analyzed in a previously established hepatocyte-specific gp130 knockout mouse model. We performed whole transcriptome analysis in isolated hepatocytes to measure tissue specific responses after proinflammatory stimulus with IL-6 across different time points. Our analyses revealed an unexpected small gene cluster that requires IL-6 stimulus for early activation. Several of the genes in this cluster are involved in different cell defense mechanisms. Thus, stressors that trigger both general stress and inflammatory responses lead to activation of a stereotypic innate cellular defense response. Furthermore, we identified a potential biomarker Lipocalin (LCN 2 for the gp130 dependent early inflammatory response. CONCLUSIONS/SIGNIFICANCE: Our findings suggest a complex network of tightly linked genes involved in the early activation of different parts of the innate immune response including acute phase proteins, complement and coagulation cascade.

  11. A cis-regulatory mutation in troponin-I of Drosophila reveals the importance of proper stoichiometry of structural proteins during muscle assembly.

    Science.gov (United States)

    Firdaus, Hena; Mohan, Jayaram; Naz, Sarwat; Arathi, Prabhashankar; Ramesh, Saraf R; Nongthomba, Upendra

    2015-05-01

    Rapid and high wing-beat frequencies achieved during insect flight are powered by the indirect flight muscles, the largest group of muscles present in the thorax. Any anomaly during the assembly and/or structural impairment of the indirect flight muscles gives rise to a flightless phenotype. Multiple mutagenesis screens in Drosophila melanogaster for defective flight behavior have led to the isolation and characterization of mutations that have been instrumental in the identification of many proteins and residues that are important for muscle assembly, function, and disease. In this article, we present a molecular-genetic characterization of a flightless mutation, flightless-H (fliH), originally designated as heldup-a (hdp-a). We show that fliH is a cis-regulatory mutation of the wings up A (wupA) gene, which codes for the troponin-I protein, one of the troponin complex proteins, involved in regulation of muscle contraction. The mutation leads to reduced levels of troponin-I transcript and protein. In addition to this, there is also coordinated reduction in transcript and protein levels of other structural protein isoforms that are part of the troponin complex. The altered transcript and protein stoichiometry ultimately culminates in unregulated acto-myosin interactions and a hypercontraction muscle phenotype. Our results shed new insights into the importance of maintaining the stoichiometry of structural proteins during muscle assembly for proper function with implications for the identification of mutations and disease phenotypes in other species, including humans.

  12. Evaluation of global sequence comparison and one-to-one FASTA local alignment in regulatory allergenicity assessment of transgenic proteins in food crops.

    Science.gov (United States)

    Song, Ping; Herman, Rod A; Kumpatla, Siva

    2014-09-01

    To address the high false positive rate using >35% identity over 80 amino acids in the regulatory assessment of transgenic proteins for potential allergenicity and the change of E-value with database size, the Needleman-Wunsch global sequence alignment and a one-to-one (1:1) local FASTA search (one protein in the target database at a time) using FASTA were evaluated by comparing proteins randomly selected from Arabidopsis, rice, corn, and soybean with known allergens in a peer-reviewed allergen database (http://www.allergenonline.org/). Compared with the approach of searching >35%/80aa+, the false positive rate measured by specificity rate for identification of true allergens was reduced by a 1:1 global sequence alignment with a cut-off threshold of ≧30% identity and a 1:1 FASTA local alignment with a cut-off E-value of ≦1.0E-09 while maintaining the same sensitivity. Hence, a 1:1 sequence comparison, especially using the FASTA local alignment tool with a biological relevant E-value of 1.0E-09 as a threshold, is recommended for the regulatory assessment of sequence identities between transgenic proteins in food crops and known allergens.

  13. Lowering of plasma phospholipid transfer protein activity by acute hyperglycaemia-induced hyperinsulinaemia in healthy men

    NARCIS (Netherlands)

    vanTol, A; Ligtenberg, JJM; Riemens, SC; vanHaeften, TW; Dullaart, RPF

    1997-01-01

    Human plasma contains two lipid transfer proteins involved in the remodelling of plasma lipoproteins: cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP). CETP mediates the transfer/exchange of cholesterylesters, triglycerides and phospholipids between high-density lip

  14. L-Alanylglutamine inhibits signaling proteins that activate protein degradation, but does not affect proteins that activate protein synthesis after an acute resistance exercise.

    Science.gov (United States)

    Wang, Wanyi; Choi, Ran Hee; Solares, Geoffrey J; Tseng, Hung-Min; Ding, Zhenping; Kim, Kyoungrae; Ivy, John L

    2015-07-01

    Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation.

  15. Systemic acute phase proteins response in calves experimentally infected with Eimeria zuernii.

    Science.gov (United States)

    Lassen, Brian; Bangoura, Berit; Lepik, Triin; Orro, Toomas

    2015-09-15

    Acute phase proteins (APPs) have been demonstrated to be useful in evaluating general health stress and diseases in cattle. Serum amyloid A (SAA) and haptoglobin (Hp) are APPs that are produced during inflammation, and likely play a role in host immunological defence against Eimeria infection and the associated intestinal tissue damage. We investigated the involvement of SAA and HP in an experimental study, including three groups of calves: a control group (group 0, n=11), and two groups infected with either 150,000 or 250,000 Eimeria zuernii oocysts (group 1 (n=11) and group 2 (n=12), respectively). The calves were monitored for 28 days and data was collected on oocyst excretion, faecal score, animal weight, and SAA and Hp serum concentrations. Generalized linear mixed models showed that the clinical symptoms, indicated by an increase in the number of oocysts in the faeces and severe diarrhoea, manifested at patency for group 1 and 2. Serum Hp and SAA levels also increased during this period. Hp appeared to be a more sensitive marker than SAA, and differences between groups 1 and 2 were observed only for Hp. Linear regression models showed a negative association between weight gain and Hp concentrations, calculated as the area under the curve (AUC) during the overall experimental period and the patency period. A similar result was seen for SAA only during the patency period. This result supports the assumption that reduced weight gain due to E. zuernii infection is an immunologically driven process that involves an increase in APPs. A random intercept regression model of oocyst shedding groups showed that calves shedding 1-500 oocysts had reduced concentrations of Hp, indicating that a different immunological reaction occurs during mild shedding of E. zuernii oocysts than during more intensive shedding. A similar model was used to examine associations between faecal scores and Hp concentrations for each group. Group 2 calves with haemorrhagic diarrhoea displayed

  16. X-ray structure of a hydroxylase-regulatory protein complex from a hydrocarbon-oxidizing multicomponent monooxygenase, Pseudomonas sp. OX1 phenol hydroxylase.

    Science.gov (United States)

    Sazinsky, Matthew H; Dunten, Pete W; McCormick, Michael S; DiDonato, Alberto; Lippard, Stephen J

    2006-12-26

    Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 A resolution. PHM binds in a canyon on one side of the (alphabetagamma)2 PHH dimer, contacting alpha-subunit helices A, E, and F approximately 12 A above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more pi-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an approximately 6 A pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O2 activation, suggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH alpha-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket

  17. Acute phase protein response during subclinical infection of pigs with H1N1 swine influenza virus.

    Science.gov (United States)

    Pomorska-Mól, Małgorzata; Markowska-Daniel, Iwona; Pejsak, Zygmunt

    2012-10-12

    In the present study acute phase proteins (APPs) responses in pigs after subclinical infection with H1N1 swine influenza virus (SwH1N1) were evaluated. Fourteen 5 weeks old, seronegative piglets, both sexes were used. Ten of them were infected intranasally with SwH1N1. C-reactive protein (CRP), haptoglobin (Hp), serum amyloid A (SAA) and pig major acute phase protein (Pig-MAP) concentrations in serum were measured using commercial ELISAs. No significant clinical signs were observed in any of the infected pigs, however, all infected animals developed specific antibodies against SwH1N1 and viral shedding was observed from 2 to 5 dpi. Only concentrations of Hp and SAA were significantly induced after infection, with mean maximum levels from days 1 to 2 post infection (dpi). The concentrations of CRP and Pig-MAP remained generally unchanged, however in half of infected pigs the concentration of CRP tended to increase at 1 dpi (but without statistical significance). The results of our study confirmed that monitoring of APPs may be useful for detection of subclinically infected pigs. The use of SAA or Hp and Pig-MAP may be a valuable in combination [i.e. Hp (increased concentration) and Pig-MAP (unchanged concentration)] to detect subclinically SIV infected pigs, or to identify pigs actually producing a large amount of virus. Additional studies need to be done in order to confirm these findings.

  18. 3T3-L1 preadipocytes exhibit heightened monocyte-chemoattractant protein-1 response to acute fatty acid exposure.

    Science.gov (United States)

    Dordevic, Aimee L; Konstantopoulos, Nicky; Cameron-Smith, David

    2014-01-01

    Preadipocytes contribute to the inflammatory responses within adipose tissue. Whilst fatty acids are known to elicit an inflammatory response within adipose tissue, the relative contribution of preadipocytes and mature adipocytes to this is yet to be determined. We aimed to examine the actions of common dietary fatty acids on the acute inflammatory and adipokine response in 3T3-L1 preadipocytes and differentiated mature adipocytes. Gene expression levels of key adipokines in 3T3-L1 preadipocytes and adipocytes were determined following incubation with palmitic acid, myristic acid or oleic acid and positive inflammatory control, lipopolysaccharide for 2 and 4 h. Inflammatory kinase signalling was assessed by analysis of nuclear factor-κB, p38-mitogen-activated protein kinase and c-jun amino-terminal kinase phosphorylation. Under basal conditions, intracellular monocyte chemoattractant protein-1 and interleukin-6 gene expression levels were increased in preadipocytes, whereas mature adipocytes expressed increased gene expression levels of leptin and adiponectin. Fatty acid exposure at 2 and 4 h increased both monocyte chemoattractant protein-1 and interleukin-6 gene expression levels in preadipocytes to greater levels than in mature adipocytes. There was an accompanying increase of inhibitor of κB-α degradation and nuclear factor-κB (p65) (Ser536) phosphorylation with fatty acid exposure in the preadipocytes only. The current study points to preadipocytes rather than the adipocytes as the contributors to both immune cell recruitment and inflammatory adipokine secretion with acute increases in fatty acids.

  19. Decreased basal chloride secretion and altered cystic fibrosis transmembrane conductance regulatory protein, Villin, GLUT5 protein expression in jejunum from leptin-deficient mice

    Directory of Open Access Journals (Sweden)

    Leung L

    2014-07-01

    Full Text Available Lana Leung, Jonathan Kang, Esa Rayyan, Ashesh Bhakta, Brennan Barrett, David Larsen, Ryan Jelinek, Justin Willey, Scott Cochran, Tom L Broderick, Layla Al-NakkashDepartment of Physiology, Arizona College of Osteopathic Medicine, Midwestern University, Glendale, AZ, USAAbstract: Patients with diabetes and obesity are at increased risk of developing disturbances in intestinal function. In this study, we characterized jejunal function in the clinically relevant leptin-deficient ob/ob mouse, a model of diabetes and obesity. We measured transepithelial short circuit current (Isc, across freshly isolated segments of jejunum from 12-week-old ob/ob and lean C57BL/6J (female and male mice. The basal Isc was significantly decreased (~30% in the ob/ob mice (66.5±5.7 µA/cm2 [n=20] (P< 0.05 compared with their lean counterparts (95.1±9.1 µA/cm2 [n=19]. Inhibition with clotrimazole (100 µM, applied bilaterally was significantly reduced in the ob/ob mice (−7.92%±3.67% [n=15] (P<0.05 compared with the lean mice (10.44%±7.92% [n=15], indicating a decreased contribution of Ca2+-activated K+ (KCa channels in the ob/ob mice. Inhibition with ouabain (100 µM, applied serosally was significantly reduced in the ob/ob mice (1.40%±3.61%, n=13 (P< 0.05 versus the lean mice (18.93%±3.76% [n=18], suggesting a potential defect in the Na+/K+-adenosine triphosphate (ATPase pump with leptin-deficiency. Expression of cystic fibrosis transmembrane conductance regulatory protein (CFTR (normalized to glyceraldehyde-3-phosphate dehydrogenase [GAPDH] was significantly decreased ~twofold (P<0.05 in the ob/ob mice compared with the leans, whilst crypt depth was unchanged. Villi length was significantly increased by ~25% (P<0.05 in the ob/ob mice compared with the leans and was associated with an increase in Villin and GLUT5 expression. GLUT2 and SGLT-1 expression were both unchanged. Our data suggests that reduced basal jejunal Isc in ob/ob mice is likely a consequence of

  20. The spike protein of severe acute respiratory syndrome (SARS) is cleaved in virus infected Vero-E6 cells

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Spike protein is one of the major structural proteins of severe acute respiratory syndrome-coronavirus. It is essential for the interaction of the virons with host cell receptors and subsequent fusion of the viral envelop with host cell membrane to allow infection. Some spike proteins of coronavirus, such as MHV, HCoV-OC43, AIBV and BcoV, are proteolytically cleaved into two subunits, S1 and S2. In contrast, TGV, FIPV and HCoV-229E are not. Many studies have shown that the cleavage of spike protein seriously affects its function. In order to investigate the maturation and proteolytic processing of the S protein of SARS CoV, we generated S1 and S2 subunit specific antibodies (Abs) as well as N, E and 3CL protein-specific Abs. Our results showed that the antibodies could efficiently and specifically bind to their corresponding proteins from E. coli expressed or lysate of SARS-CoV infected Vero-E6 cells by Western blot analysis. Furthermore, the anti-S 1 and S2 Abs were proved to be capable of binding to SARS CoV under electron microscope observation. When S2 Ab was used to perform immune precipitation with lysate of SARS-CoV infected cells, a cleaved S2 fragment was detected with S2-specific mAb by Western blot analysis. The data demonstrated that the cleavage of S protein was observed in the lysate, indicating that proteolytic processing of S protein is present in host cells.

  1. Early prognosis of survival or death after a recent stroke by blood levels of acute-phase proteins.

    Science.gov (United States)

    Ionescu, D A; Haţegan, D; Jipescu, I; Steinbruch, L; Ghiţescu, M

    1991-01-01

    From 129 patients with a recent stroke 105 survived and 24 died within 3 weeks from stroke-onset. At around 40 hours after the latter, the blood-levels of the acute-phase proteins ceruloplasmin and albumin did not forecast the death of the respective patients, but, in contradistinction, the level of fibrinogen was significantly higher in those who eventually died, than in those who survived. Therefore, a higher level of fibrinogen could be a risk-factor for death after stroke.

  2. Evaluation of high mobility group box 1 protein as a presurgical diagnostic marker reflecting the severity of acute appendicitis

    Directory of Open Access Journals (Sweden)

    Wu Chuanxin

    2012-09-01

    Full Text Available Abstract Objectives To validate the role of high mobility group box-1(HMGB1 in diagnosis of acute appendicitis (AA with different pathological severity. Methods According to the pathologically diagnosis, 150 patients underwent appendectomies between Jan. 2007 and Dec, 2010 were divided into acute simple, acute suppurative and acute gangrenous appendicitis as group 1, 2 and 3, respectively. Each patient group contains 50 sex and age matched cases to make comparison with 50 healthy volunteers. The mRNA and protein expression levels of serum HMGB1 were determined by real-time quantitative PCR and enzyme linked immunosorbent assay (ELISA. Serum High-sensitivity C-reactive protein (hs-CRP levels were determined by rate nephelometric immunoassay. Results In comparison with health volunteers, relative HMGB1 mRNA levels in group 1, 2 and 3 were significantly increased 3.05 ± 0.51,8.33 ± 0.75 and 13.74 ± 1.09 folds, reflecting a tendency of augmented severity. In accordance, serum protein levels of HMGB1 were 10.97 ± 1.64, 14.42 ± 1.56 and 18.08 ± 2.41 ng/ml in 3 patient groups, which are significantly higher than that of healthy volunteers’ 5.47 ± 0.73 ng/ml. hs-CRP levels were 12.85 ± 3.41, 21.04 ± 1.98 and 31.07 ± 5.46 ng/ml in 3 patients groups compared with 2.06 ± 0.77 ng/ml in controls. The concentrations of HMGB1 and hs-CRP were both positively correlated with disease severity. Conclusion Serum HMGB1 constitutes as a valuable marker in diagnosis of AA. Positively correlated with hs-CRP level, mRNA and protein expression of HMGB1 to a certain extent reflected the severity of AA.

  3. Joint Influence of Protein Supplements, Soft Drinks and Extreme Physical Activity on the Development of Acute Renal Failure and Hypokalaemia.

    Science.gov (United States)

    Djordjevic, S; Kitic, D; Kostic, M; Apostolovic, B; Brankovic, S; Ciric, I M; Velickovic-Radovanovic, R

    2015-11-13

    We present a case of a 33-year old man who complained of weakness, fever and decreased urinating. A personal history revealed a consumption of creatine, protein supplements, soft drinks containing caffeine and stevia, and extreme physical activity which included lifting of heavy weights. The patient developed anuria, uraemia, fatigue, rhabdomyolysis and paradoxical hypokalaemia. After the patient had seven successive dialysis treatments, normal kidney function was restored. The report presents the first case of acute renal failure followed by hypokalaemia due to the combined action of the excessive consumption of supplements, soft drinks with stevia and caffeine, and extreme physical activity.

  4. Expression of the gonococcal global regulatory protein Fur and genes encompassing the Fur and iron regulon during in vitro and in vivo infection in women.

    Science.gov (United States)

    Agarwal, Sarika; Sebastian, Shite; Szmigielski, Borys; Rice, Peter A; Genco, Caroline A

    2008-05-01

    The ferric uptake regulatory protein, Fur, functions as a global regulatory protein of gene transcription in the mucosal pathogen Neisseria gonorrhoeae. We have shown previously that several N. gonorrhoeae Fur-repressed genes are expressed in vivo during mucosal gonococcal infection in men, which suggests that this organism infects in an iron-limited environment and that Fur is expressed under these conditions. In this study we have demonstrated expression of the gonococcal fur gene in vitro, in human cervical epithelial cells, and in specimens from female subjects with uncomplicated gonococcal infection. In vitro studies confirmed that the expression of the gonococcal fur gene was repressed during growth under iron-replete growth conditions but that a basal level of the protein was maintained. Using GFP transcriptional fusions constructed from specific Fur binding sequences within the fur promoter/operator region, we determined that this operator region was functional during N. gonorrhoeae infection of cervical epithelial cells. Furthermore, reverse transcription-PCR analysis, as well as microarray analysis, using a custom Neisseria Fur and iron regulon microarray revealed that several Fur- and iron-regulated genes were expressed during N. gonorrhoeae infection of cervical epithelial cells. Microarray analysis of specimens obtained from female subjects with uncomplicated gonococcal infection corroborated our in vitro findings and point toward a key role of gonococcal Fur- and iron-regulated genes in gonococcal disease.

  5. Occupational exposure levels of bioaerosol components are associated with serum levels of the acute phase protein Serum Amyloid A in greenhouse workers

    DEFF Research Database (Denmark)

    Madsen, Anne Mette; Thilsing, Trine; Bælum, Jesper;

    2016-01-01

    to elevated levels of bioaerosols. The objective of this study is to assess whether greenhouse workers personal exposure to bioaerosol components was associated with serum levels of the acute phase proteins Serum Amyloid A (SAA) and C-reactive protein (CRP). METHODS: SAA and CRP levels were determined...

  6. Procalcitonin and C-reactive protein-based decision tree model for distinguishing PFAPA flares from acute infections

    Directory of Open Access Journals (Sweden)

    Barbara Kraszewska-Głomba

    2016-03-01

    Full Text Available As no specific laboratory test has been identified, PFAPA (periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis remains a diagnosis of exclusion. We searched for a practical use of procalcitonin (PCT and C-reactive protein (CRP in distinguishing PFAPA attacks from acute bacterial and viral infections. Levels of PCT and CRP were measured in 38 patients with PFAPA and 81 children diagnosed with an acute bacterial (n=42 or viral (n=39 infection. Statistical analysis with the use of the C4.5 algorithm resulted in the following decision tree: viral infection if CRP≤19.1 mg/L; otherwise for cases with CRP>19.1 mg/L: bacterial infection if PCT>0.65ng/mL, PFAPA if PCT≤0.65 ng/mL. The model was tested using a 10-fold cross validation and in an independent test cohort (n=30, the rule’s overall accuracy was 76.4% and 90% respectively. Although limited by a small sample size, the obtained decision tree might present a potential diagnostic tool for distinguishing PFAPA flares from acute infections when interpreted cautiously and with reference to the clinical context.

  7. Procalcitonin and C-reactive protein-based decision tree model for distinguishing PFAPA flares from acute infections.

    Science.gov (United States)

    Kraszewska-Głomba, Barbara; Szymańska-Toczek, Zofia; Szenborn, Leszek

    2016-03-10

    As no specific laboratory test has been identified, PFAPA (periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis) remains a diagnosis of exclusion. We searched for a practical use of procalcitonin (PCT) and C-reactive protein (CRP) in distinguishing PFAPA attacks from acute bacterial and viral infections. Levels of PCT and CRP were measured in 38 patients with PFAPA and 81 children diagnosed with an acute bacterial (n=42) or viral (n=39) infection. Statistical analysis with the use of the C4.5 algorithm resulted in the following decision tree: viral infection if CRP≤19.1 mg/L; otherwise for cases with CRP>19.1 mg/L: bacterial infection if PCT>0.65ng/mL, PFAPA if PCT≤0.65 ng/mL. The model was tested using a 10-fold cross validation and in an independent test cohort (n=30), the rule's overall accuracy was 76.4% and 90% respectively. Although limited by a small sample size, the obtained decision tree might present a potential diagnostic tool for distinguishing PFAPA flares from acute infections when interpreted cautiously and with reference to the clinical context.

  8. ACUTE PHASE PROTEIN RESPONSES IN MICE INFECTED WITH RIVER WATER CONTAMINATED BY PASTEURELLA MULTOCIDA TYPE B: 2

    Directory of Open Access Journals (Sweden)

    Mohammed Muqdad Khaleel

    2013-01-01

    Full Text Available Animals are predisposed to infections through varieties of ways which activate the innate immune systems at the initial phase of acute infections. Hemorrhagic Septicemia (HS is a devastating septicemic disease of cattle and buffaloes caused by a particular serotypes of Pasteurella multocida and serotype B: 2 is the most important cause of the disease in Asia. Therefore, the present study aims to investigate on acute phase protein responses in mice infected with river water contaminated by Pasteurella multocida type B: 2. Five infected mice were placed in each tank containing river water for 24, 48 and 72 h. The groups comprise of five mice each made up of the control, intraperitoneal, oral and the aerosol routes. There were increased concentrations of Haptoglobin (Hp in the mice inoculated with infected river water kept for 24 h intraperitoneally (p<0.0454 relative to the control, oral and the aerosol routes. There was significant increased concentration of Serum Amyloid A (SAA in the mice that were inoculated with infected river water kept for 72 h intraperitoneally (p<0.0020 compared to control, orally and the aerosol routes. The intraperitoneal route though most effective in response but practically not feasible for vaccine administration in larger animals and the oral routes was inadequate in eliciting acute phase response in the present study of mice model. Therefore, the aerosol routes could perhaps be a readily available route for effective vaccine administration and heightened immunity in animals considering the progressive responses of APPs through this route.

  9. Relationship Between the Hypersensitive c-Reactive Protein (hs-CRP) Level and the Prognosis of Acute Brainstem Infarction.

    Science.gov (United States)

    Zhang, Xia; Huang, Wen-Juan; Yu, Zhi-Gang

    2015-05-01

    The objective of this study is to explore the relationship between the hypersensitive c-reactive protein (hs-CRP) level and the prognosis of acute brainstem infarction. Serum levels of hs-CRP were measured in 68 patients with acute brainstem infarction 72 h after disease onset. The hs-CRP levels in the U.S. National Institutes of Health Stroke Scale (NIHSS) score group and in the modified RANKIN scale (mRS) score group were compared. The independent risk factors of brainstem infarction were analyzed using Logistic binary regression. The hs-CRP level was significantly higher in the group with NIHSS >5 compared with the one with NIHSS ≤ 5 (P = 0.004). In the group with mRS > 2, the age, smoking history, and blood glucose level were significantly higher than those in the group with mRS ≤ 2 (P hs-CRP level was significantly higher (P = 0.001). Age and hs-CRP level were the independent prognostic factors of the brainstem infarction. The serum hs-CRP level is closely related with the severity and prognosis of brainstem infarction, and is an independent risk factor of acute brainstem infarction.

  10. Diagnostic utility of C-reactive Protein combined with brain natriuretic peptide in acute pulmonary edema: a cross sectional study

    Directory of Open Access Journals (Sweden)

    Murakami Junji

    2011-06-01

    Full Text Available Abstract Introduction Discriminating acute lung injury (ALI or acute respiratory distress syndrome (ARDS from cardiogenic pulmonary edema (CPE using the plasma level of brain natriuretic peptide (BNP alone remains controversial. The aim of this study was to determine the diagnostic utility of combination measurements of BNP and C-reactive protein (CRP in critically ill patients with pulmonary edema. Methods This was a cross-sectional study. BNP and CRP data from 147 patients who presented to the emergency department due to acute respiratory failure with bilateral pulmonary infiltrates were analyzed. Results There were 53 patients with ALI/ARDS, 71 with CPE, and 23 with mixed edema. Median BNP and CRP levels were 202 (interquartile range 95-439 pg/mL and 119 (62-165 mg/L in ALI/ARDS, and 691 (416-1,194 pg/mL (p Conclusions Measurement of CRP is useful as well as that of BNP for distinguishing ALI/ARDS from CPE. Furthermore, a combination of BNP and CRP can provide higher accuracy for the diagnosis.

  11. Potential effects of calcium binding protein S100A12 on severity evaluation and curative effect of severe acute pancreatitis.

    Science.gov (United States)

    Feng, Zhang; Yinchu, Zhan; Yinsheng, Shi; Fengqing, Wu; Xiaoyang, Zhou; Jin, Li; Xiaofei, Gao

    2015-02-01

    Severe acute pancreatitis is a life threatening disease with a high rate of mortality, but its treatments are still controversial. The purpose of this study is to investigate the potential effects of calcium binding protein S100A12 on severity evaluation and curative effect of severe acute pancreatitis induced by caerulein and lipopolysaccharide in mice. Intraperitoneal injection of 50 μg/kg caerulein for seven times (every interval time was an hour) and intraperitoneal injection of 10 mg/kg lipopolysaccharide for once to establish acute pancreatitis mice models. One hundred sixty specific pathogen-free imprinting control region (ICR) female mice were randomly divided into the control group (group A, normal saline), the mild group (group B, caerulein), the severe group (group C, caerulein + lipopolysaccharide), and the intervention group (group D, S100A12 recombinant antibodies + caerulein + lipopolysaccharide); each group had 40 mice. We sampled the blood at 8, 12, and 24 h after the beginning of building animal models. In each period of time, we respectively detected the serum S100A12, amylase (AMY), C-reactive protein (CRP), interleukin (IL-1β, IL-6), and tumor necrosis factor (TNF-α) levels. In addition, we observed and scored the pancreas and lungs histopathology of the mice. In each same period of time compared with group C, serum AMY, CRP, IL-1β, IL-6, TNF-α levels of group D were significantly decreased (p < 0.05). In each same period of time compared with group B and group C, serum S100A12 concentration of group D was significantly decreased (p < 0.05), and the pancreas and lungs histopathology were also much improved. These observations demonstrate that S100A12 recombinant antibodies were able to significantly reduce the severity of acute pancreatitis induced by caerulein and lipopolysaccharide in mice. Serum S100A12 may serve as a useful marker for disease severity and curative effect in mice with severe acute pancreatitis.

  12. Expanding the archaellum regulatory network - the eukaryotic protein kinases ArnC and ArnD influence motility of Sulfolobus acidocaldarius.

    Science.gov (United States)

    Hoffmann, Lena; Schummer, Andreas; Reimann, Julia; Haurat, Maria F; Wilson, Amanda J; Beeby, Morgan; Warscheid, Bettina; Albers, Sonja-V

    2017-02-01

    Expression of the archaellum, the archaeal-type IV pilus-like rotating motility structure is upregulated under nutrient limitation. This is controlled by a network of regulators, called the archaellum regulatory network (arn). Several of the components of this network in Sulfolobus acidocaldarius can be phosphorylated, and the deletion of the phosphatase PP2A results in strongly increased motility during starvation, indicating a role for phosphorylation in the regulation of motility. Analysis of the motility of different protein kinase deletion strains revealed that deletion of saci_0965, saci_1181, and saci_1193 resulted in reduced motility, whereas the deletion of saci_1694 resulted in hypermotility. Here ArnC (Saci_1193) and ArnD (Saci_1694) are characterized. Purified ArnC and ArnD phosphorylate serine and threonine residues in the C-terminus of the repressor ArnB. arnC is upregulated in starvation medium, whereas arnD is constitutively expressed. However, while differences in the expression and levels of flaB were observed in the ΔarnD strain during growth under rich conditions, under nutrient limiting conditions the ΔarnC and ΔarnD strains showed no large differences in the expression levels of the archaellum or of the studied regulators. This suggests that next to the regulation via the archaellum regulatory network additional regulatory mechanisms of expression and/or activity of the archaellum exist.

  13. The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies

    Directory of Open Access Journals (Sweden)

    Burt Austin

    2009-07-01

    Full Text Available Abstract Background Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae. Results We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae. Conclusion We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression.

  14. AGGRESSIVE CHEMOTHERAPY FOR ACUTE-LEUKEMIA FREQUENTLY CAUSES INTESTINAL PROTEIN LEAKAGE

    NARCIS (Netherlands)

    DAENEN, S; MUSKIET, FAJ; MARRINK, J; HALIE, MR

    1991-01-01

    Cytostatic drugs are known to produce disturbances in intestinal absorption of carbohydrates. To further explore the gastrointestinal (GI) toxicity of cytostatic therapy, 37 patients with acute leukaemia were investigated during and/or after remission induction courses by the use of the differential

  15. Protein C activation during the initial phase of experimental acute pancreatitis in the rabbit

    DEFF Research Database (Denmark)

    Ottesen, L H; Bladbjerg, E-M; Osman, M;

    2000-01-01

    BACKGROUND: Disturbances of coagulation and fibrinolysis are well-known systemic effects of acute necrotising pancreatitis (ANP). The purpose of this experimental study was to evaluate the initial events in the haemostatic activation during ANP in an animal model with relevance to the human...

  16. Targeted proteomics as a tool for porcine acute phase proteins measurements

    DEFF Research Database (Denmark)

    Marco-Ramell, Anna; Bassols, Anna; Bislev, Stine Lønnerup

    2013-01-01

    ) and there is recent evidence that they can be also considered as welfare markers (Giannetto et al., 2011). During an acute stress situation, the levels of some interleukins (IL-1β, IL-6 and TNF-α) are increased (Elenkov and Chrousos, 2002) and consequently the concentration of the APPs varies dramatically. In pigs...

  17. Preventing phosphorylation of sterol regulatory element-binding protein 1a by MAP-kinases protects mice from fatty liver and visceral obesity.

    Science.gov (United States)

    Kotzka, Jorg; Knebel, Birgit; Haas, Jutta; Kremer, Lorena; Jacob, Sylvia; Hartwig, Sonja; Nitzgen, Ulrike; Muller-Wieland, Dirk

    2012-01-01

    The transcription factor sterol regulatory element binding protein (SREBP)-1a plays a pivotal role in lipid metabolism. Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK). Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK) or p38 stress activated MAP kinases. Serine 117 is also the major phosphorylation site in SREBP-1a for JNK. In contrast to that the major phosphorylation sites of p38 MAPK family are serine 63 and threonine 426. Functional analyses reveal that phosphorylation of SREBP-1a does not alter protein/DNA interaction. The identified phosphorylation sites are specific for both kinase families also in cellular context. To provide direct evidence that phosphorylation of SREBP-1a is a regulatory principle of biological and clinical relevance, we generated transgenic mice expressing mature transcriptionally active N-terminal domain of human SREBP-1a variant lacking all identified phosphorylaton sites designed as alb-SREBP-1aΔP and wild type SREBP-1a designed as alb-SREBP-1a liver specific under control of the albumin promoter and a liver specific enhancer. In contrast to alb-SREBP-1a mice the phosphorylation-deficient mice develop no enlarged fatty livers under normocaloric conditions. Phenotypical examination reveales a massive accumulation of adipose tissue in alb-SREBP-1a but not in the phosphorylation deficient alb-SREBP-1aΔP mice. Moreover, preventing phosphorylation of SREBP-1a protects mice also from dyslipidemia. In conclusion, phosphorylation of SREBP-1a by ERK, JNK and p38 MAPK-families resembles a biological principle and plays a significant role, in vivo.

  18. Preventing phosphorylation of sterol regulatory element-binding protein 1a by MAP-kinases protects mice from fatty liver and visceral obesity.

    Directory of Open Access Journals (Sweden)

    Jorg Kotzka

    Full Text Available The transcription factor sterol regulatory element binding protein (SREBP-1a plays a pivotal role in lipid metabolism. Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK. Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK or p38 stress activated MAP kinases. Serine 117 is also the major phosphorylation site in SREBP-1a for JNK. In contrast to that the major phosphorylation sites of p38 MAPK family are serine 63 and threonine 426. Functional analyses reveal that phosphorylation of SREBP-1a does not alter protein/DNA interaction. The identified phosphorylation sites are specific for both kinase families also in cellular context. To provide direct evidence that phosphorylation of SREBP-1a is a regulatory principle of biological and clinical relevance, we generated transgenic mice expressing mature transcriptionally active N-terminal domain of human SREBP-1a variant lacking all identified phosphorylaton sites designed as alb-SREBP-1aΔP and wild type SREBP-1a designed as alb-SREBP-1a liver specific under control of the albumin promoter and a liver specific enhancer. In contrast to alb-SREBP-1a mice the phosphorylation-deficient mice develop no enlarged fatty livers under normocaloric conditions. Phenotypical examination reveales a massive accumulation of adipose tissue in alb-SREBP-1a but not in the phosphorylation deficient alb-SREBP-1aΔP mice. Moreover, preventing phosphorylation of SREBP-1a protects mice also from dyslipidemia. In conclusion, phosphorylation of SREBP-1a by ERK, JNK and p38 MAPK-families resembles a biological principle and plays a significant role, in vivo.

  19. Induction of Heat Shock Protein 72 in RGCs of Rat Acute Glaucoma Model after Heat Stress or Zinc Administration

    Institute of Scientific and Technical Information of China (English)

    Guoping Qing; Xuanchu Duan; Youqin Jiang

    2004-01-01

    Purpose :To investigate the dynamics of heat shock protein 72 (HSP72) expression in retinal ganglion cells (RGCs) in rat model of acute glaucoma treated with heat stress or intraperitoneal injection of zinc sulfate.Methods: Twenty-seven male Wistar rats were used to make acute glaucoma models. Five others served as normal control. Acute glaucoma models were made by intracameral irrigation in the right eyes with balanced salt saline (BSS) at 102 mmHg for 2 hours. Nine model rats were killed at different intervals after intracameral irrigation without treatment, which served as damage control. Ten were treated with heat stress 40℃~42℃, and 8 were used for zinc sulfate administration 2 days posterior to intracameral irrigation.Treated model rats were sacrificed at designed intervals after treatment. Right eyes were enucleated immediately, and the retinas were dissected for Western blot.Results: No HSP72 was found in RGCs of normal Wistar rats. In damage control group,slight HSP72 was detected during 6~36 hours posterior to intracameral irrigation. HSP72was detected significantly expressed in RGCs of both heat shock group and zinc sulfate group. But the dynamics of HSP72 production were quite different in these two treated groups. In heat shock group, HSP72 appeared at the sixth hour after treatment, and increased gradually until its peak production emerged at the 48th hour. HSP72 vanished 8days later after treatment. In zinc sulfate group, HSP72 expression began 24 hours later after zinc administration, and reached its highest level at the 72th hour posterior to treatment. HSP72 expression then decreased slowly, and disappeared 21 days later after treatment.Conclusion:HSP72 can be induced in RGCs of rat acute glaucoma models with heat stress or zinc sulfate adddministration. But the dynamics of the HSP72 induction in those two groups were quite different. Eye Science 2004;20:30-33.

  20. Cloning, Expression, Purification and Crystallization of NHR3 Domain from Acute Myelogenous Leukemia-related Protein AML1-ETO

    Institute of Scientific and Technical Information of China (English)

    Hai-Tao YANG; Dong-Hui WU; Xiao-Yu XUE; Wen-Xue LIANG; Xiao-Yu MIAO; Hai PANG; Sai-Juan CHEN

    2004-01-01

    The t(8;21)translocation is one of the most frequent chromosome abnormalities in acute myeloid leukemia.This transtocation creates a fusion between the acute myelogenous leukemia 1(AML1,a transcription factor)gene on chromosome 21 and the eight-twenty-one(ETO,a zinc finger nuclear protein)gene on chromosome 8,leading to the repression of certain AML 1 target genes.We cloned NHR3 domain coding fragment into vector pGEX-6p-1 using PCR and obtained recombinant plasmid pGEX-6p-1-NHR3,which can be induced to stably overexpress fusion protein in E.coli.Through the purification on GST affinity chromatography column and PreScission protease cleavage,a large amount of NHR3 protein with high purity was obtained.In order to avoid possible interference of some strong negative charged molecules,NHR3 protein was further purified by Mono Q anion exchange chromatography.The NHR3 crystals were grown with hanging drop/vapor diffusion method and the first crystals appeared after four weeks at 18 ℃in 0.2 M Tris-sodium citrate dihydrate,0.1 M sodium cacodylate,pH 6.5,and 30% iso-propanol(V/V).ESI mass spectrum showed that the molecular weight of this domain was in agreement with its primary structure sequence prediction,and circular dichroism spectral data(190-250 nm)showed that NHR3 had a well-defined secondary structure of 25.9% α-helix,23.2% random coil and 50.9% turn,which was consistent with GOV4 software prediction.

  1. Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii

    OpenAIRE

    2014-01-01

    Triacylglycerol (TG) synthesis is induced for energy and carbon storage in algal cells under nitrogen(N)-starved conditions, and helps prevent reactive oxygen species (ROS) production through fatty acid synthesis that consumes excessive reducing power. Here, the regulatory mechanism for the TG content in sulfur(S)-starved cells of Chlamydomonas reinhardtii was examined, in comparison to that in N- or phosphorus(P)-starved cells. S- and N- starved cells exhibited markedly increased TG contents...

  2. Correlative mRNA and protein expression of middle and inner ear inflammatory cytokines during mouse acute otitis media.

    Science.gov (United States)

    Trune, Dennis R; Kempton, Beth; Hausman, Frances A; Larrain, Barbara E; MacArthur, Carol J

    2015-08-01

    Although the inner ear has long been reported to be susceptible to middle ear disease, little is known of the inflammatory mechanisms that might cause permanent sensorineural hearing loss. Recent studies have shown inner ear tissues are capable of expressing inflammatory cytokines during otitis media. However, little quantitative information is available concerning cytokine gene expression in the inner ear and the protein products that result. Therefore, this study was conducted of mouse middle and inner ear during acute otitis media to measure the relationship between inflammatory cytokine genes and their protein products with quantitative RT-PCR and ELISA, respectively. Balb/c mice were inoculated transtympanically with heat-killed Haemophilus influenzae and middle and inner ear tissues collected for either quantitative RT-PCR microarrays or ELISA multiplex arrays. mRNA for several cytokine genes was significantly increased in both the middle and inner ear at 6 h. In the inner ear, these included MIP-2 (448 fold), IL-6 (126 fold), IL-1β (7.8 fold), IL-10 (10.7 fold), TNFα (1.8 fold), and IL-1α (1.5 fold). The 24 h samples showed a similar pattern of gene expression, although generally at lower levels. In parallel, the ELISA showed the related cytokines were present in the inner ear at concentrations higher by 2-122 fold higher at 18 h, declining slightly from there at 24 h. Immunohistochemistry with antibodies to a number of these cytokines demonstrated they occurred in greater amounts in the inner ear tissues. These findings demonstrate considerable inflammatory gene expression and gene products in the inner ear following acute otitis media. These higher cytokine levels suggest one potential mechanism for the permanent hearing loss seen in some cases of acute and chronic otitis media.

  3. Elevation of intact and proteolytic fragments of acute phase proteins constitutes the earliest systemic antiviral response in HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Holger B Kramer

    2010-05-01

    Full Text Available The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, beta-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT, termed virus inhibitory peptide (VIRIP, was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.

  4. Individuals with hematological malignancies before undergoing chemotherapy present oxidative stress parameters and acute phase proteins correlated with nutritional status.

    Science.gov (United States)

    Camargo, Carolina de Quadros; Borges, Dayanne da Silva; de Oliveira, Paula Fernanda; Chagas, Thayz Rodrigues; Del Moral, Joanita Angela Gonzaga; Durigon, Giovanna Steffanello; Dias, Bruno Vieira; Vieira, André Guedes; Gaspareto, Patrick; Trindade, Erasmo Benício Santos de Moraes; Nunes, Everson Araújo

    2015-01-01

    Hematological malignancies present abnormal blood cells that may have altered functions. This study aimed to evaluate nutritional status, acute phase proteins, parameters of cell's functionality, and oxidative stress of patients with hematological malignancies, providing a representation of these variables at diagnosis, comparisons between leukemias and lymphomas and establishing correlations. Nutritional status, C-reactive protein (CRP), albumin, phagocytic capacity and superoxide anion production of mononuclear cells, lipid peroxidation and catalase activity in plasma were evaluated in 16 untreated subjects. Main diagnosis was acute leukemia (n = 9) and median body mass index (BMI) indicated overweight (25.6 kg/m(2)). Median albumin was below (3.2 g/dL) and CRP above (37.45 mg/L) the reference values. Albumin was inversely correlated with BMI (r = -0.53). Most patients were overweight before the beginning of treatment and had a high CRP/albumin ratio, which may indicate a nutrition inflammatory risk. BMI values correlated positively with lipid peroxidation and catalase activity. A strong correlation between catalase activity and lipid peroxidation was found (r = 0.75). Besides the elevated BMI, these patients also have elevated CRP values and unexpected relations between nutritional status and albumin, reinforcing the need for nutritional counseling during the course of chemotherapy, especially considering the correlations between oxidative stress parameters and nutritional status evidenced here.

  5. Effect of acute endurance and resistance exercise on endocrine hormones directly related to lipolysis and muscle protein synthesis in adult individuals with obesity: a review.

    OpenAIRE

    2012-01-01

    In subjects with obesity, the implementation of long-term exercise intervention increases lean tissue mass and lowers adipose tissue mass. However, data indicate a blunted lipolytic response, and/or skeletal muscle protein synthesis, when subjects with obesity are exposed to acute endurance or resistance exercise, respectively. Therefore, subjects with obesity seem to display a suboptimal physiological response to acute exercise stimuli. It might be hypothesized that hormonal disturb...

  6. Levels of Regulatory Proteins Associated With Cell Proliferation in Endometria From Untreated Patients Having Polycystic Ovarian Syndrome With and Without Endometrial Hyperplasia.

    Science.gov (United States)

    Bacallao, K; Plaza-Parrochia, F; Cerda, A; Gabler, F; Romero, C; Vantman, D; Vega, M

    2016-02-01

    Polycystic ovarian syndrome (PCOS) has been associated with endometrial hyperplasia and cancer. The aim of this study was to establish whether the expression of proliferation regulatory proteins in the endometria of patients having PCOS, with or without hyperplasia, differs from control women. Control endometria (CE), patients having PCOS without and with endometrial hyperplasia (PCOSE and HPCOSE, respectively), and that of women with endometrial hyperplasia (HE) were used. The phosphorylated estrogen receptor form (pERα), similar to mother against decapentaplegic (SMAD) 2, SMAD3, and SMAD4, vascular epithelial growth factor (VEGF), and phosphorylated SMAD (pSMAD) 2 and pSMAD3 were detected by immunohistochemistry or Western blot. The results show higher levels of pERα in HE versus CE (P hyperplasia and cancer, whereas changes observed in SMAD proteins support the differential origin of the pathologies of HPCOSE and HE.

  7. Lysosomal-associated Transmembrane Protein 4B (LAPTM4B) Decreases Transforming Growth Factor β1 (TGF-β1) Production in Human Regulatory T Cells.

    Science.gov (United States)

    Huygens, Caroline; Liénart, Stéphanie; Dedobbeleer, Olivier; Stockis, Julie; Gauthy, Emilie; Coulie, Pierre G; Lucas, Sophie

    2015-08-14

    Production of active TGF-β1 is one mechanism by which human regulatory T cells (Tregs) suppress immune responses. This production is regulated by glycoprotein A repetitions predominant (GARP), a transmembrane protein present on stimulated Tregs but not on other T lymphocytes (Th and CTLs). GARP forms disulfide bonds with proTGF-β1, favors its cleavage into latent inactive TGF-β1, induces the secretion and surface presentation of GARP·latent TGF-β1 complexes, and is required for activation of the cytokine in Tregs. We explored whether additional Treg-specific protein(s) associated with GARP·TGF-β1 complexes regulate TGF-β1 production in Tregs. We searched for such proteins by yeast two-hybrid assay, using GARP as a bait to screen a human Treg cDNA library. We identified lysosomal-associated transmembrane protein 4B (LAPTM4B), which interacts with GARP in mammalian cells and is expressed at higher levels in Tregs than in Th cells. LAPTM4B decreases cleavage of proTGF-β1, secretion of soluble latent TGF-β1, and surface presentation of GARP·TGF-β1 complexes by Tregs but does not contribute to TGF-β1 activation. Therefore, LAPTM4B binds to GARP and is a negative regulator of TGF-β1 production in human Tregs. It may play a role in the control of immune responses by decreasing Treg immunosuppression.

  8. Magnolol causes alterations in the cell cycle in androgen insensitive human prostate cancer cells in vitro by affecting expression of key cell cycle regulatory proteins.

    Science.gov (United States)

    McKeown, Brendan T; McDougall, Luke; Catalli, Adriana; Hurta, Robert A R

    2014-01-01

    Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 μM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16(INK4a), p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.

  9. Inhibitory Effects of NO-Fluvastatin on Proliferation of Human Lens Epithelial Cells in vitro by Modulating Cell Cycle Regulatory Proteins

    Institute of Scientific and Technical Information of China (English)

    Zhi WANG; Ruiying GAO; Qianqian SHI; Yukan HUANG; Wen CHEN; Kaiying SHI

    2008-01-01

    Summary: The effects of NO-Fluvastatin on proliferation of human lens epithelial cells (HLECs) and the action mechanism were investigated. Cell proliferation was assessed by MTT assay. Cell cycle was analyzed by flow cytometry. The expression of cell cycle regulatory proteins CyclinE mRNA and P21wafl mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). MTT staining colorimetry showed that HLECs proliferation was markedly inhibited by NO-Fluvastatin and the effect was dependently related to time (24, 48 and 72 h) and dosage (1, 5 and 20 μmol/L). Flow cytometry revealed that NO-Fluvastatin could significantly block HLECs in the G0/G1 phase, resulting in the increased cells in the G0G1 phase and decreased in the S phase (P<0.05). RT-PCR showed that NO-Fluvastatin could obviously inhibit the CyclinE mRNA expression and induce the P21wafl mRNA expression as compared with the negative control groups (P<0.05). This experiment suggested that NO-Fluvastatin could suppress the proliferation of HLECs by regulating cell cycle regulatory proteins (inhibiting the expression of CyclinE mRNA and inducing the expression of P21wafl mRNA), resulting in the arrest of HLECs in the G0/G1 phase, which can offer theory basis for NO-Fluvastatin in treating posterior capsular opacification in clinic practice.

  10. Regulatory circuit for responses of nitrogen catabolic gene expression to the GLN3 and DAL80 proteins and nitrogen catabolite repression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Daugherty, J R; Rai, R; el Berry, H M; Cooper, T G

    1993-01-01

    We demonstrate that expression of the UGA1, CAN1, GAP1, PUT1, PUT2, PUT4, and DAL4 genes is sensitive to nitrogen catabolite repression. The expression of all these genes, with the exception of UGA1 and PUT2, also required a functional GLN3 protein. In addition, GLN3 protein was required for expression of the DAL1, DAL2, DAL7, GDH1, and GDH2 genes. The UGA1, CAN1, GAP1, and DAL4 genes markedly increased their expression when the DAL80 locus, encoding a negative regulatory element, was disrupted. Expression of the GDH1, PUT1, PUT2, and PUT4 genes also responded to DAL80 disruption, but much more modestly. Expression of GLN1 and GDH2 exhibited parallel responses to the provision of asparagine and glutamine as nitrogen sources but did not follow the regulatory responses noted above for the nitrogen catabolic genes such as DAL5. Steady-state mRNA levels of both genes did not significantly decrease when glutamine was provided as nitrogen source but were lowered by the provision of asparagine. They also did not respond to disruption of DAL80.

  11. Variations at regulatory regions of the milk protein genes are associated with milk traits and coagulation properties in the Sarda sheep.

    Science.gov (United States)

    Noce, A; Pazzola, M; Dettori, M L; Amills, M; Castelló, A; Cecchinato, A; Bittante, G; Vacca, G M

    2016-12-01

    Regulatory variation at the ovine casein genes could have important effects on the composition and coagulation properties of milk. Herewith, we have partially resequenced the promoters and the 3'-UTR of the four casein genes in 25 Sarda sheep. Alignment of these sequences allowed us to identify a total of 29 SNPs. This level of polymorphism (one SNP every 250 bp) is remarkably high if compared with SNP densities estimated in human genic regions (approximately one SNP per bp). The 29 SNPs identified in our resequencing experiment, plus three previously reported SNPs mapping to the lactalbumin, alpha (LALBA) and β-lactoglobulin (BLG, also known as progestagen-associated endometrial protein, PAEP) genes, were genotyped with a multiplex TaqMan Open Array Real-Time PCR assay in 760 Sarda sheep with records for milk composition and coagulation properties. Association analysis revealed the existence of significant associations of CSN1S2 and CSN3 genotypes with milk protein and casein contents. Moreover, genotypes at CSN1S1 were significantly associated with rennet coagulation time, curd firming time and curd firmness, whereas CSN2 was associated with curd firming time. These results suggest that SNPs mapping to the promoters and 3'-UTRs of ovine casein genes may exert regulatory effects on gene expression and that they could be used for improving sheep milk quality and technological traits at the population level through marker assisted selection.

  12. Aging is Associated with Impaired Renal Function, INF-gamma Induced Inflammation and with Alterations in Iron Regulatory Proteins Gene Expression

    Science.gov (United States)

    Costa, Elísio; Fernandes, João; Ribeiro, Sandra; Sereno, José; Garrido, Patrícia; Rocha-Pereira, Petronila; Coimbra, Susana; Catarino, Cristina; Belo, Luís; Bronze-da-Rocha, Elsa; Vala, Helena; Alves, Rui; Reis, Flávio; Santos-Silva, Alice

    2014-01-01

    Our aim was to contribute to a better understanding of the pathophysiology of anemia in elderly, by studying how aging affects renal function, iron metabolism, erythropoiesis and the inflammatory response, using an experimental animal model. The study was performed in male Wistar, a group of young rats with 2 months age and an old one with 18 months age. Old rats presented a significant higher urea, creatinine, interferon (INF)-gamma, ferritin and soluble transferrin receptor serum levels, as well as increased counts of reticulocytes and RDW. In addition, these rats showed significant lower erythropoietin (EPO) and iron serum levels. Concerning gene expression of iron regulatory proteins, old rats presented significantly higher mRNA levels of hepcidin (Hamp), transferrin (TF), transferrin receptor 2 (TfR2) and hemojuvelin (HJV); divalent metal transporter 1 (DMT1) mRNA levels were significantly higher in duodenal tissue; EPO gene expression was significantly higher in liver and lower in kidney, and the expression of the EPOR was significantly higher in both liver and kidney. Our results showed that aging is associated with impaired renal function, which could be in turn related with the inflammatory process and with a decline in EPO renal production. Moreover, we also propose that aging may be associated with INF-gamma-induced inflammation and with alterations upon iron regulatory proteins gene expression. PMID:25489488

  13. Alterations in oxidant/antioxidant balance, high-mobility group box 1 protein and acute phase response in cross-bred suckling piglets suffering from rotaviral enteritis.

    Science.gov (United States)

    Kumar De, Ujjwal; Mukherjee, Reena; Nandi, Sukdeb; Patel, Bhimnere Hanumatnagouda Manjunatha; Dimri, Umesh; Ravishankar, Chintu; Verma, Ashok Kumar

    2014-10-01

    Rotaviral enteritis has emerged as a major cause of morbidity and mortality in piglets during their post-natal life. The present study was carried out to examine high-mobility group box 1 (HMGB1) protein, acute phase response and oxidative stress indices in the serum of suckling piglets suffering from enteritis with or without association of porcine group A rotavirus infection. The present investigation utilized 23 clinical cases with signs of acute enteritis and 12 more healthy piglets of a similar age group as control animals. Out of 23 enteritis cases, 12 cases were found to be positive for porcine group A rotavirus infection as confirmed by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for group A rotavirus, and the rest were found negative. The acute enteritis cases in piglets were associated with an elevated level of HMGB1 protein and serum haptoglobin and ceruloplasmin suggestive of an acute phase response. Among the oxidative stress indices, the concentrations of malondialdehyde (MDA) and nitric oxide (NO) in serum were significantly increased. A pronounced drop of total antioxidant capacity and the activity of antioxidant enzymes such as catalase and superoxide dismutase in the serum of piglets suffering from acute enteritis compared to healthy ones were also noticed. The alterations in HMGB1 protein, acute phase response and oxidative stress indices were more pronounced in cases with the involvement of porcine rotavirus as compared to rotavirus-negative cases. It is concluded that HMGB1 protein, markers of oxidative stress and acute phase proteins might play an important role in the aetiopathogenesis of porcine diarrhoea caused by rotavirus and might be true markers in diagnosing the conditions leading to the extension of the prompt and effective therapeutic care.

  14. IgG responses to Pneumococcal and Haemophilus influenzae protein antigens are not impaired in children with a history of recurrent acute otitis media.

    Directory of Open Access Journals (Sweden)

    Selma P Wiertsema

    Full Text Available BACKGROUND: Vaccines including conserved antigens from Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi have the potential to reduce the burden of acute otitis media. Little is known about the antibody response to such antigens in young children with recurrent acute otitis media, however, it has been suggested antibody production may be impaired in these children. METHODS: We measured serum IgG levels against 4 pneumococcal (PspA1, PspA 2, CbpA and Ply and 3 NTHi (P4, P6 and PD proteins in a cross-sectional study of 172 children under 3 years of age with a history of recurrent acute otitis media (median 7 episodes, requiring ventilation tube insertion and 63 healthy age-matched controls, using a newly developed multiplex bead assay. RESULTS: Children with a history of recurrent acute otitis media had significantly higher geometric mean serum IgG levels against NTHi proteins P4, P6 and PD compared with healthy controls, whereas there was no difference in antibody levels against pneumococcal protein antigens. In both children with and without a history of acute otitis media, antibody levels increased with age and were significantly higher in children colonised with S. pneumoniae or NTHi compared with children that were not colonised. CONCLUSIONS: Proteins from S. pneumoniae and NTHi induce serum IgG in children with a history of acute otitis media. The mechanisms in which proteins induce immunity and potential protection requires further investigation but the dogma of impaired antibody responses in children with recurrent acute otitis media should be reconsidered.

  15. Acute phase proteins in dogs naturally infected with the Giant Kidney Worm (Dioctophyme renale)

    DEFF Research Database (Denmark)

    Schmidt, Elizabeth M. S.; Kjelgaard-Hansen, Mads; Thomas, Funmilola

    2016-01-01

    Background: Dioctophyme renale is a nematode parasite of dogs, usually found in the right kidney, causing severe damage to the renal parenchyma. Objectives: The objective was to evaluate the acute phase response in dogs naturally infected with this Giant Kidney Worm and the possible effects...... of parasites and APP concentrations. Conclusion: There is a particular acute phase response profile in dogs with kidney worm infection. Nephrectomy induced a short-term inflammatory process....... and Friedman test were used for multiple comparisons; the Wilcoxon-signed rank test was used to compare variables, and Spearman’s rho rank test was used to assess the correlation between the number of parasites recovered from the dogs and the APP concentration. Results: Forty-five parasites were recovered from...

  16. Identification of target proteins of mangiferin in mice with acute lung injury using functionalized magnetic microspheres based on click chemistry.

    Science.gov (United States)

    Wang, Jiajia; Nie, Yan; Li, Yunjuan; Hou, Yuanyuan; Zhao, Wei; Deng, Jiagang; Wang, Peng George; Bai, Gang

    2015-11-18

    Prevention of the occurrence and development of inflammation is a vital therapeutic strategy for treating acute lung injury (ALI). Increasing evidence has shown that a wealth of ingredients from natural foods and plants have potential anti-inflammatory activity. In the present study, mangiferin, a natural C-glucosyl xanthone that is primarily obtained from the peels and kernels of mango fruits and the bark of the Mangifera indica L. tree, alleviated the inflammatory responses in lipopolysaccharide (LPS)-induced ALI mice. Mangiferin-modified magnetic microspheres (MMs) were developed on the basis of click chemistry to capture the target proteins of mangiferin. Mass spectrometry and molecular docking identified 70 kDa heat-shock protein 5 (Hspa5) and tyrosine 3-monooxygenase (Ywhae) as mangiferin-binding proteins. Furthermore, an enzyme-linked immunosorbent assay (ELISA) indicated that mangiferin exerted its anti-inflammatory effect by binding Hspa5 and Ywhae to suppress downstream mitogen-activated protein kinase (MAPK) signaling pathways. Thoroughly revealing the mechanism and function of mangiferin will contribute to the development and utilization of agricultural resources from M. indica L.

  17. Acute phase protein concentrations in serum and milk from healthy cows, cows with clinical mastitis and cows with extramammary inflammatory conditions

    DEFF Research Database (Denmark)

    Nielsen, B.H.; Jacobsen, S.; Andersen, P.H.

    2004-01-01

    The concentrations of the two acute phase proteins, serum amyloid A and haptoglobin, in serum and milk were compared in 10 cows with clinical mastitis, 11 cows with extramammary inflammatory conditions and 10 clinically healthy control cows. The concentrations of both acute phase proteins were...... higher in the serum and milk of the cows with mastitis than in the cows in the other two groups. Four of the cows with extramammary inflammatory conditions had serum amyloid A concentrations in serum above 100 mug/ml, but negligible concentrations in milk, indicating that a pathogen must be present...... in the mammary gland for serum amyloid A to accumulate in milk. The acute phase protein concentrations in milk increased significantly with increasing somatic cell count, suggesting that they may be indicators of the severity of an infection....

  18. The diagnostic and prognostic importance of oxidative stress biomarkers and acute phase proteins in Urinary Tract Infection (UTI) in camels.

    Science.gov (United States)

    El-Deeb, Wael M; Buczinski, Sébastien

    2015-01-01

    The present study aimed to investigate the diagnostic and prognostic importance of oxidative stress biomarkers and acute phase proteins in urinary tract infection (UTI) in camels. We describe the clinical, bacteriological and biochemical findings in 89 camels. Blood and urine samples from diseased (n = 74) and control camels (n = 15) were submitted to laboratory investigations. The urine analysis revealed high number of RBCS and pus cells. The concentrations of serum and erythrocytic malondialdehyde (sMDA & eMDA), Haptoglobin (Hp), serum amyloid A (SAA), Ceruloplasmin (Cp), fibrinogen (Fb), albumin, globulin and interleukin 6 (IL-6) were higher in diseased camels when compared to healthy ones. Catalase, super oxide dismutase and glutathione levels were lower in diseased camels when compared with control group. Forty one of 74 camels with UTI were successfully treated. The levels of malondialdehyde, catalase, super oxide dismutase, glutathione, Hp, SAA, Fb, total protein, globulin and IL-6 were associated with the odds of treatment failure. The MDA showed a great sensitivity (Se) and specificity (Sp) in predicting treatment failure (Se 85%/Sp 100%) as well as the SAA (Se 92%/Sp 87%) and globulin levels (Se 85%/Sp 100%) when using the cutoffs that maximizes the sum of Se + Sp. Multivariate logistic regression analysis revealed that two models had a high accuracy to predict failure with the first model including sex, sMDA and Hp as covariates (area under the receiver operating characteristic curve (AUC) = 0.92) and a second model using sex, SAA and Hp (AUC = 0.89). Conclusively, the oxidative stress biomarkers and acute phase proteins could be used as diagnostic and prognostic biomarkers in camel UTI management. Efforts should be forced to investigate such biomarkers in other species with UTI.

  19. Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes

    Science.gov (United States)

    Ledesma-García, Laura; Sánchez-Azqueta, Ana; Medina, Milagros; Reyes-Ramírez, Francisca; Santero, Eduardo

    2016-01-01

    Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H–ThnA4–ThnA3–ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H–ThnA4–ThnA3–ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction. PMID:27030382

  20. Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

    Directory of Open Access Journals (Sweden)

    H. Carvalho

    2008-04-01

    Full Text Available Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1 plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.

  1. Effect of acute endurance and resistance exercise on endocrine hormones directly related to lipolysis and skeletal muscle protein synthesis in adult individuals with obesity.

    Science.gov (United States)

    Hansen, Dominique; Meeusen, Romain; Mullens, Annelies; Dendale, Paul

    2012-05-01

    In subjects with obesity, the implementation of long-term exercise intervention increases lean tissue mass and lowers adipose tissue mass. However, data indicate a blunted lipolytic response, and/or skeletal muscle protein synthesis, when subjects with obesity are exposed to acute endurance or resistance exercise, respectively. Therefore, subjects with obesity seem to display a suboptimal physiological response to acute exercise stimuli. It might be hypothesized that hormonal disturbances contribute, at least in part, to these abnormal physiological reactions in the obese. This review discusses the impact of acute endurance and resistance exercise on endocrine hormones directly related to lipolysis and/or skeletal muscle protein synthesis (insulin, [nor]epinephrine, cortisol, growth hormone, testosterone, triiodothyronine, atrial natriuretic peptide, insulin-like growth factor-1), as well as the impact of long-term endurance and resistance exercise intervention on these hormonal responses to acute endurance and resistance exercise. In the obese, some endocrinological disturbances during acute endurance and resistance exercise have been identified: a blunted blood growth hormone, atrial natriuretic peptide and epinephrine release, and greater cortisol and insulin release. These hormonal disturbances might contribute to a suppressed lipolytic response, and/or suppressed skeletal muscle protein synthesis, as a result of acute endurance or resistance exercise, respectively. In subjects with obesity, the impact of acute endurance and resistance exercise on other endocrine hormones (norepinephrine, testosterone, triiodothyronine, insulin-like growth factor-1) remains elusive. Furthermore, whether long-term endurance and resistance exercise intervention might reverse these hormonal disturbances during acute endurance and resistance exercise in these individuals remains unknown.

  2. Time elapsed after transplantation influences the relationship between the number of regulatory T cells in lung allograft biopsies and subsequent acute rejection episodes

    DEFF Research Database (Denmark)

    Krustrup, Dorrit; Iversen, Martin; Martinussen, Torben;

    2014-01-01

    scored for acute rejection according to the ISHLT criteria (A0-A4) and immunohistochemically stained with antibodies against FoxP3. Results: There was a tendency for a decrease in the number of Tregs/mm2 with time. However, the previous levels of Tregs/mm2 did not have any significant effect on future...

  3. Heterogeneity in the acute control of vascular protein synthesis in vivo.

    Science.gov (United States)

    Beaucage, Pierre; Yamaguchi, Nobuharu; Larivière, Richard; Moreau, Pierre

    2003-01-01

    In response to both hemodynamic and neurohumoral changes, the cardiovascular system remodels and this process could contribute to end organ damage. The aim of this study was to determine the early in vivo interactions between 3 systems known to contribute to vascular hypertrophic remodeling, in conduit and resistance arteries. Exogenous angiotensin II, norepinephrine and endothelin 1 administration elevated protein synthesis in the aorta and in small mesenteric arteries. In small arteries, the effect of angiotensin II was blocked by angiotensin II type 1, alpha-adrenergic and endothelin receptor antagonists, while only the alpha-adrenergic and endothelin receptor antagonists inhibited the effect of norepinephrine. Moreover, only the endothelin receptor antagonist significantly blunted the effect of exogenous endothelin on protein synthesis. In the aorta, the stimulation of angiotensin II on protein synthesis was also inhibited by the 3 antagonists. However, only the alpha-adrenoceptor antagonist blunted the response to norepinephrine, and the 3 antagonists prevented the endothelin-induced elevation of protein synthesis. The blood pressure effects of the drugs did not correlate with their capacity to stimulate or inhibit vascular protein synthesis. In conclusion, interactions in the control of protein synthesis are heterogeneous along the vascular tree. In small arteries, the interaction is linear with endothelin as the downstream effector. In the aorta, the local sympathetic nervous system appears to control protein synthesis. The heterogeneity in downstream effectors should be considered in studies investigating signaling events related to protein synthesis, which is used as an early marker of hypertrophy.

  4. The role of breast cancer resistance protein in acute lymphoblastic leukemia

    NARCIS (Netherlands)

    Plasschaert, SLA; van der Kolk, D.M.; de Bout, ESJM; Kamps, WA; Morisaki, K; Bates, SE; Scheffer, GL; Scheper, RJ; Vellenga, E; de Vries, EGE

    2003-01-01

    Purpose: Overexpression of the transporter ABCG2, also known as breast cancer resistance protein and mitoxantrone resistance protein, can confer resistance to a variety of cytostatic drugs, such as mitoxantrone, topotecan, doxorubicin, and daunorubicin. This study analyzes the ABCG2 expression and a

  5. The Lipid Transfer Protein StarD7: Structure, Function, and Regulation

    OpenAIRE

    Susana Genti-Raimondi; Panzetta-Dutari, Graciela M.; Sofía Angeletti; Viviana Rena; Jésica Flores-Martin

    2013-01-01

    The steroidogenic acute regulatory (StAR) protein-related lipid transfer (START) domain proteins constitute a family of evolutionarily conserved and widely expressed proteins that have been implicated in lipid transport, metabolism, and signaling. The 15 well-characterized mammalian START domain-containing proteins are grouped into six subfamilies. The START domain containing 7 mRNA encodes StarD7, a member of the StarD2/phosphatidylcholine transfer protein (PCTP) subfamily, which was first i...

  6. Phylogenetic divergence of CD47 interactions with human signal regulatory protein alpha reveals locus of species specificity. Implications for the binding site.

    Science.gov (United States)

    Subramanian, Shyamsundar; Boder, Eric T; Discher, Dennis E

    2007-01-19

    Cell-cell interactions between ubiquitously expressed integrin-associated protein (CD47) and its counterreceptor signal regulatory protein (SIRPalpha) on phagocytes regulate a wide range of adhesive signaling processes, including the inhibition of phagocytosis as documented in mice. We show that CD47-SIRPalpha binding interactions are different between mice and humans, and we exploit phylogenetic divergence to identify the species-specific binding locus on the immunoglobulin domain of human CD47. All of the studies are conducted in the physiological context of membrane protein display on Chinese hamster ovary (CHO) cells. Novel quantitative flow cytometry analyses with CD47-green fluorescent protein and soluble human SIRPalpha as a probe show that neither human CD47 nor SIRPalpha requires glycosylation for interaction. Human CD47-expressing CHO cells spread rapidly on SIRPalpha-coated glass surfaces, correlating well with the spreading of primary human T cells. In contrast, CHO cells expressing mouse CD47 spread minimally and show equally weak binding to soluble human SIRPalpha. Further phylogenetic analyses and multisite substitutions of the CD47 Ig domain show that human to cow mutation of a cluster of seven residues on adjacent strands near the middle of the domain decreases the association constant for human SIRPalpha to about one-third that of human CD47. Direct tests of cell-cell adhesion between human monocytes and CD47-displaying CHO cells affirm the species specificity as well as the importance of the newly identified binding locus in cell-cell interactions.

  7. Radiometric ligand binding assay for C-reactive protein. Complexed C-reactive protein is not detectable in acute phase serum.

    Science.gov (United States)

    De Beer, F C; Shine, B; Pepys, M B

    1982-10-01

    A radiometric ligand binding assay for human C-reactive protein (CRP) was established using pneumococcal C polysaccharide (CPS) coupled to magnetizable cellulose particles as the solid phase ligand. Competition for binding to the solid phase between 125I-CRP and unlabelled CRP permitted detection of 30 micrograms/l of CRP and the precise assay of concentrations up to 3000 micrograms/l. Identical results were obtained when the assay was used to quantitate isolated pure CRP and pure CRP added to normal human serum. However in vitro addition of known ligands for CRP to acute phase serum resulted in lowering of the apparent CRP concentration in this assay and addition of as little as 1 microgram/l of free CPS or 1 mg/l of lecithin was demonstrable in this way. A combination of the ligand binding assay and the standard electroimmunoassay for CRP was therefore used to test acute phase sera for the presence of CRP complexed in vitro. No evidence of complexed CRP was detected among sera containing between 1-319 mg/l of CRP from patients with Hodgkin's disease (10), rheumatoid arthritis (10), Crohn's disease (19) and various microbial infections (11), including six with subacute bacterial endocarditis. Since it is likely that CRP does form complexes with its ligands in the plasma these results suggest that complexed CRP is rapidly cleared from the circulation.

  8. Preserved skeletal muscle protein anabolic response to acute exercise and protein intake in well-treated rheumatoid arthritis patients

    DEFF Research Database (Denmark)

    Mikkelsen, Ulla Ramer; Dideriksen, Kasper; Andersen, Mads Bisgaard

    2015-01-01

    of genes related to inflammatory signaling, myogenesis and muscle growth/atrophy were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: CRP was significantly higher in the RA patients (2.25 (0.50) mg/l) than in controls (1.07 (0.25) mg/l; p = 0.038) and so was TNF...... similar in RA vs. CON, however, expression of the genes coding for TNF-α, myogenin and HGF1 were more responsive to exercise in RA patients than in CON. CONCLUSIONS: The study demonstrates that muscle protein synthesis rate and muscle gene expression can be stimulated by protein intake alone...... and anabolic signaling response in patients with RA compared to healthy controls. METHODS: Thirteen RA patients (age range 34-84 years; diagnosed for 1-32 years, median 8 years) were individually matched with 13 healthy controls for gender, age, BMI and activity level (CON). Plasma levels of C-reactive protein...

  9. HY-Specific Induced Regulatory T Cells Display High Specificity and Efficacy in the Prevention of Acute Graft-versus-Host Disease

    OpenAIRE

    Li, Jun; Heinrichs, Jessica; Haarberg, Kelley; Semple, Kenrick; Veerapathran, Anandharaman; Liu, Chen; Anasetti, Claudio; Yu, Xue-Zhong

    2015-01-01

    Naturally derived regulatory T cells (nTregs) may prevent graft-versus-host disease (GVHD) while preserving graft-versus-leukemia (GVL) activity. However, clinical application of nTregs has been severely hampered by their scarce availability and non-selectivity. To overcome these limitations, we took alternative approaches to generate Ag-specific induced Tregs (iTregs) and tested their efficacy and selectivity in the prevention of GVHD in pre-clinical models of bone marrow transplantation (BM...

  10. Proteins involved on TGF-β pathway are up-regulated during the acute phase of experimental Chagas disease.

    Science.gov (United States)

    Ferreira, Roberto Rodrigues; de Souza, Elen Mello; de Oliveira, Fabiane Loiola; Ferrão, Patrícia Mello; Gomes, Leonardo Henrique Ferreira; Mendonça-Lima, Leila; Meuser-Batista, Marcelo; Bailly, Sabine; Feige, Jean Jacques; de Araujo-Jorge, Tania Cremonini; Waghabi, Mariana Caldas

    2016-05-01

    Studies developed by our group in the last years have shown the involvement of TGF-β in acute and chronic Chagas heart disease, with elevated plasma levels and activated TGF-β cell signaling pathway as remarkable features of patients in the advanced stages of this disease, when high levels of cardiac fibrosis is present. Imbalance in synthesis and degradation of extracellular matrix components is the basis of pathological fibrosis and TGF-β is considered as one of the key regulators of this process. In the present study, we investigated the activity of the TGF-β signaling pathway, including receptors and signaling proteins activation in the heart of animals experimentally infected with Trypanosoma cruzi during the period that mimics the acute phase of Chagas disease. We observed that T. cruzi-infected animals presented increased expression of TGF-β receptors. Overexpression of receptors was followed by an increased phosphorylation of Smad2/3, p38 and ERK. Furthermore, we correlated these activities with cellular factors involved in the fibrotic process induced by TGF-β. We observed that the expression of collagen I, fibronectin and CTGF were increased in the heart of infected animals on day 15 post-infection. Correlated with the increased TGF-β activity in the heart, we found that serum levels of total TGF-β were significantly higher during acute infection. Taken together, our data suggest that the commitment of the heart associates with increased activity of TGF-β pathway and expression of its main components. Our results, confirm the importance of this cytokine in the development and maintenance of cardiac damage caused by T. cruzi infection.

  11. Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum

    Directory of Open Access Journals (Sweden)

    Ana ePalomino

    2014-03-01

    Full Text Available Growing awareness of cerebellar involvement in addiction is based on the cerebellum’s intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression (CB1 receptors and enzymes that produce (DAGLα/β and NAPE-PLD and degrade (MAGL and FAAH eCB were altered. In addition, we analyzed the gene expression of relevant components of the glutamate signaling system (glutamate synthesizing enzymes LGA and KGA, mGluR3/5 metabotropic receptors, and NR1/2A/2B/2C-NMDA and GluR1/2/3/4-AMPA ionotropic receptor subunits and the gene expression of tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamine biosynthesis, because noradrenergic terminals innervate the cerebellar cortex. Results indicated that acute cocaine exposure decreased DAGLα expression, suggesting a down-regulation of 2-AG production, as well as gene expression of TH, KGA, mGluR3 and all ionotropic receptor subunits analyzed in the cerebellum. The acquisition of conditioned locomotion and sensitization after repeated cocaine exposure were associated with an increased NAPE-PLD/FAAH ratio, suggesting enhanced anandamide production, and a decreased DAGLβ/MAGL ratio, suggesting decreased 2-AG generation. Repeated cocaine also increased LGA gene expression but had no effect on glutamate receptors. These findings indicate that acute cocaine modulates the expression of the eCB and glutamate systems. Repeated cocaine results in normalization of glutamate receptor expression, although sustained changes in eCB is observed. We suggest that cocaine-induced alterations to cerebellar eCB should be considered when analyzing the adaptations imposed by psychostimulants that

  12. Acute high-altitude hypoxic brain injury Identification of ten differential proteins

    Institute of Scientific and Technical Information of China (English)

    Jianyu Li; Yuting Qi; Hui Liu; Ying Cui; Li Zhang; Haiying Gong; Yaxiao Li; Lingzhi Li; Yongliang Zhang

    2013-01-01

    Hypobaric hypoxia can cause severe brain damage and mitochondrial dysfunction, and is involved in hypoxic brain in