Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P56851 (epididymal)
 11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

SNAP-25 (synaptosomal-associated protein 25), syntaxin and synaptobrevin are the three SNARE [soluble NSF attachment protein receptor (where NSF = N-ethylmaleimide-sensitive fusion protein)] proteins that form the core complex involved in synaptic vesicle docking and subsequent fusion with the target membrane. The present study is aimed at understanding the mechanisms of fusion of vesicles carrying glucose transporter proteins with the plasma membrane in human insulin-responsive tissues. It describes the isolation and characterization of cDNA molecules encoding SNAP-25 A and B isoforms, syntaxin 4 and synaptobrevins (also known as vehicle-associated membrane proteins) from two major human insulin-responsive tissues, skeletal muscle and fat. The DNA and deduced amino acid sequences of SNAP-25 revealed perfect identity with the previously reported human neural SNAP-25 A and B isoforms. Our results indicate the presence of both isoforms both in insulin-responsive tissues and in in vitro cultured 3T3-L1 cells, but suggest a differential pattern of gene expression: isoform A is the major species in adipose tissue, and isoform B is the major species in skeletal muscle. The presence of SNAP-25 protein in 3T3-L1 cells was demonstrated by immunofluorescence microscopy using an anti-SNAP-25 monoclonal antibody. Immunoprecipitation experiments using the same monoclonal antibody also revealed the presence of SNAP-25 protein in plasma membrane fractions from rat epididymal fat pads. The syntaxin 4-encoding region from skeletal muscle contains five nucleotide differences from the previously reported placental cDNA sequence, two of which result in amino acid changes: Asp-174 to Glu and Val-269 to Ala. The synaptobrevin 1 cDNA from skeletal muscle contains two nucleotide differences when compared with the corresponding clone from neural tissues, one of which is silent and the other resulting in the amino acid change Thr-102 to Ala. The cDNA sequence of the protein from fat is identical with that of human synaptobrevin 1 from neural tissues. Furthermore, we have confirmed the presence of syntaxin 4 in fat and of synaptobrevin 2 in skeletal muscle by PCR amplification and Southern hybridization analysis. Using the yeast two-hybrid system, an interaction was observed between the full-length cytoplasmic domains of syntaxin 4 and synaptobrevin 2, a vesicle membrane SNARE previously shown by others to be associated with vesicles carrying the GLUT4 glucose transporter protein, but no interaction was seen with synaptobrevin 1. Flow cytometry of low-density microsomes isolated from fat cells was used to demonstrate the binding of syntaxin 4 to a subset of vesicles carrying GLUT4 protein; whereas SNAP-25 on its own bound poorly to these vesicles, the syntaxin 4-SNAP-25 complex gave a strong interaction.
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PMID:Insulin-responsive tissues contain the core complex protein SNAP-25 (synaptosomal-associated protein 25) A and B isoforms in addition to syntaxin 4 and synaptobrevins 1 and 2. 876 Mar 87

A protein designated as BE-20 was purified from cauda epididymal fluid of the rabbit by preparative polyacrylamide gel electrophoresis and HPLC on a mono Q HR5/5 anion exchange column. The purified protein migrated with an estimated Mt of 20,000 when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequence of the N-terminus of the BE-20 protein was determined. The initial eight amino acid residues were His-Gly-Ala-Asp-Lys-Pro-Gly-Val. The corresponding 23 mer oligonucleotide (5'-CATGGCGCTGACAAGCCTGGGGT-3') was synthesized and used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The purified BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. The BE-20 cDNA had 78.5% identity in 479 bp overlap with human epididymis-specific HE4 cDNA. The amino acid sequences of the initial 30 amino acid residues of the N-terminus of the purified protein and the deduced polypeptides were as follows: N-His-Gly-Ala-Asp-Lys-Pro-Gly-Val-Cys-Pro-Gln-Leu-Ser-Ala-Asp-Leu-Asn-Cy s- Thr-Gln-Asp-Cys-Arg-Ala-Asp-Gln-Asp-Cys-Ala-Glu. The deduced polypeptide contained 16 cysteine residues and had partial sequence homology with proteins belonging to the four-disulfide core family of extracellular proteinase inhibitors. The BE-20 protein may play a role in sperm maturation and/or capacitation.
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PMID:Identification of a rabbit epididymal protein gene. 888 63

cAMP is important for the initiation of mammalian sperm motility. Previously we established that a type II cAMP-dependent protein kinase is tightly associated with the fibrous sheath of rat sperm. This unique cytoskeletal structure surrounds the 9+2 axonemal network in the principal piece of the flagellum. Association of the kinase to the fibrous sheath is mediated via its regulatory subunit, RII. An RII-binding overlay procedure was used to document that RII could specifically associate with fibrous sheath polypeptides of 120 and 80 kDa. In this study, we report the cloning of a rat testis-specific, developmentally regulated, RII-binding protein (TAKAP-80). A 1.2-kb cDNA clone, isolated by screening a rat testis expression library with 32P-labeled RII, hybridized to a 1.8-kb mRNA transcript present exclusively in testis. This transcript appeared at detectable levels at 30 days after birth. Over the next 10 days the mRNA levels increased greatly. This time interval corresponds to the initiation of spermiogenesis. The complete nucleotide sequence of TAKAP-80 cDNA was obtained by polymerase chain reaction and contained a continuous open reading frame of 502 amino acids. The deduced amino acid sequence showed a clear demarcation of charged and hydrophobic amino acid residues. Amino acids 1-147 of the protein contained 45% charged residues, with lysine and arginine predominating. Similarly, amino acids 268-502 also contained a high percentage of charged amino acids (35%). In contrast, amino acids 148-267 were mostly hydrophobic and contained clusters of a repeating PXXP motif where X was predominantly valine and alanine or sometimes proline. The 1.2-kb cDNA clone was inserted into the pRSET vector and expressed as a His6 tag fusion protein in Escherichia coli. The recombinant protein was soluble and bound RIIalpha, RIIbeta and type IIalpha holoenzyme by the RII-binding overlay procedure. Deletion analysis revealed that the high-affinity interaction site for RII was contained within amino acids 258-378 of TAKAP-80. Antibodies prepared against the fusion protein recognized an 80-kDa protein present in the urea-insoluble particulate fraction of rat testis and in purified fibrous sheath preparations isolated from rat epididymal sperm. Levels of the 80-kDa immunoreactive protein were significantly higher in mature (60 days old) compared with immature (30 days old) rat testis, correlating with the mRNA levels.
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PMID:Cloning and characterization of a testis-specific, developmentally regulated A-kinase-anchoring protein (TAKAP-80) present on the fibrous sheath of rat sperm. 920 34

Using mechanical and chemical dissection methods, fibrous sheath was isolated both from normal ejaculated human spermatozoa and from rabbit cauda epididymal spermatozoa. The same techniques did not produce a pure preparation of fibrous sheath from ejaculated rabbit spermatozoa, suggesting that further cross-linking and stabilization of sperm structures occurs in response to components of the seminal plasma. The isolation procedures were monitored by phase contrast microscopy and the purity of the fibrous sheath was verified by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human fibrous sheath revealed at least 14 protein bands of which the most intensely stained were of molecular weight 84, 72, 66.2, 57, 32 and 28.5 kDa. The rabbit fibrous sheath revealed at least 10 protein bands, of which the most intensely stained were 35.2, 32.7 and 28.5 kDa. The amino acid composition of the purified fibrous sheath from human and rabbit spermatozoa was similar, being high in aspartic acid and/or asparagine and glutamic acid and/or glutamine, serine, alanine, leucine, lysine and glycine, but low in histidine, tyrosine and isoleucine. This composition is similar to that reported for the rat and suggests that mammalian sperm tail fibrous sheaths are composed of similar types of proteins, although there are apparent differences in protein components between species.
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PMID:Isolation and characterization of human and rabbit sperm tail fibrous sheath. 923 58

Studies were carried out to analyze the cryoprotecting efficacy of several amino acids by use of a chemically defined synthetic medium (modified Ringer's solution) and goat cauda epididymal sperm as the model system. Motile goat cauda sperm dispersed in the synthetic medium were subjected to a freezing protocol in a computer-controlled bio-freezer, cooling 0.25 degrees C x min(-1) to 5 degrees C, 5 degrees C x min(-1) to -20 degrees C, and 20 degrees C x min(-1) to -100 degrees C, prior to being plunged into liquid nitrogen. In the absence of amino acids, sperm cells completely lost their flagellar motility. Of all the amino acids tested, l-alanine showed maximal cryoprotection potential. l-Alanine at 135 mM offered optimum cryoprotection potential: recovery of sperm forward motility and total motility were 14 +/- 2% and 19 +/- 2%, respectively. l-Glutamine, l-proline, and glycine at optimum concentration (100-150 mM) cryopreserved approx. 11-17% total motility of the sperm cells, whereas amino acids such as l-arginine, l-lysine, and l-histidine offered little cryoprotection (0-5%) to the cells. Increasing the amino acid concentration beyond the optimum level sharply decreased the recovery of the sperm motility, which therefore showed a biphasic cryoprotection profile. Addition of amino acids enhanced (approx. 7-10%) the cryoprotection efficacy of the well-known cryoprotectants glycerol and a combination of glycerol and dimethyl sulfoxide. The presence of glycerol caused a marked reduction (from 100-150 mM to 20-70 mM levels) in the optimal cryoprotective concentration of the amino acids. The combined cryoprotecting action of glycerol, dimethyl sulfoxide, and amino acids provided motility recovery as high as 52%. The observation that amino acids and dimethyl sulfoxide had an additive effect in augmenting the cryoprotecting potential of glycerol suggests that the mechanism of their action is different from that of glycerol. This cocktail of cryoprotectants may be useful for cryopreservation of semen of various species.
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PMID:Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system. 1133 86

This study was conducted to test the hypothesis that dietary supplementation of arginine, the physiologic precursor of nitric oxide (NO), reduces fat mass in the Zucker diabetic fatty (ZDF) rat, a genetically obese animal model of type-II diabetes mellitus. Male ZDF rats, 9 wk old, were pair-fed Purina 5008 diet and received drinking water containing arginine-HCl (1.51%) or alanine (2.55%, isonitrogenous control) for 10 wk. Serum concentrations of arginine and NO(x) (oxidation products of NO) were 261 and 70% higher, respectively, in arginine-supplemented rats than in control rats. The body weights of arginine-treated rats were 6, 10, and 16% lower at wk 4, 7, and 10 after the treatment initiation, respectively, compared with control rats. Arginine supplementation reduced the weight of abdominal (retroperitoneal) and epididymal adipose tissues (45 and 25%, respectively) as well as serum concentrations of glucose (25%), triglycerides (23%), FFA (27%), homocysteine (26%), dimethylarginines (18-21%), and leptin (32%). The arginine treatment enhanced NO production (71-85%), lipolysis (22-24%), and the oxidation of glucose (34-36%) and octanoate (40-43%) in abdominal and epididymal adipose tissues. Results of the microarray analysis indicated that arginine supplementation increased adipose tissue expression of key genes responsible for fatty acid and glucose oxidation: NO synthase-1 (145%), heme oxygenase-3 (789%), AMP-activated protein kinase (123%), and peroxisome proliferator-activated receptor gamma coactivator-1alpha (500%). The induction of these genes was verified by real-time RT-PCR analysis. In sum, arginine treatment may provide a potentially novel and useful means to enhance NO synthesis and reduce fat mass in obese subjects with type-II diabetes mellitus.
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PMID:Dietary L-arginine supplementation reduces fat mass in Zucker diabetic fatty rats. 1579 23

The isoflavones--genistein and daidzein -- compounds found in high concentrations in soy play an important role in prevention of many diseases and affect some metabolic pathways. In the performed experiment it was demonstrated that genistein (5mg/kg b.w.) administered intragastrically for three days to male Wistar rats substantially diminished blood leptin level. Studies with isolated rat adipocytes revealed that this phytoestrogen strongly restricted leptin secretion from these cells. These effects were not accompanied by any changes in leptin gene expression in adipocytes. Daidzein-- an analogue of genistein -- used at similar concentrations did not affect blood leptin concentration, leptin secretion and expression of its gene. To determine the influence of genistein and daidzein on leptin release, adipocytes isolated from the epididymal fat tissue were incubated for 2h in Krebs--Ringer buffer. Leptin secretion stimulated by glucose with insulin was significantly diminished by genistein (0.25--1mM). This effect of genistein may arise from several aspects of its action in adipocytes documented in the literature such as the inhibition of glucose transport and metabolism, the attenuation of insulin signalling, the inhibition of cAMP phosphodiesterase and the stimulation of lipolysis. However, the bypassing of the restrictive action of genistein on glucose transport and glycolysis (by the use of alanine instead of glucose) and on insulin action (by the use of nicotinic acid) was not sufficient to restore leptin secretion from isolated adipocytes. It was also demonstrated that the restriction of the stimulatory influence of genistein on cAMP/protein kinase A (PKA) pathway (by the inhibition of PKA activity) did not improve leptin release. Results obtained in our experiments point at the restriction of glucose metabolism following formation of pyruvate as the pivotal reason of the inhibitory action of genistein on leptin release.
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PMID:Genistein restricts leptin secretion from rat adipocytes. 1597 Apr 40

The partitioning of whole body carbon flux between fat and lean compartments affects body composition. We hypothesized that it is possible to simultaneously determine whole body carbon (energy) balance and the dynamics of lipids and proteins in specific tissues in vivo. Growing C57BL/6J mice fed a high-fat low-carbohydrate diet were injected with a bolus of "doubly labeled" water (i.e., (2)H2O and H2(18)O). The rate of CO2 production was determined from the difference between the elimination rates of 2H and 18O from body water. The rates of synthesis and degradation of triglycerides extracted from epididymal fat pads and of proteins extracted from heart muscle were determined by mathematically modeling the 2H labeling of triglyceride-bound glycerol and protein-bound alanine, respectively. We found that mice were in positive carbon balance (approximately 20% retention per day) and accumulated lipid in epididymal fat pads (approximately 9 micromol triglyceride accumulated per day). This is consistent with the fact that mice were studied during a period of growth. Modeling the 2H labeling of triglycerides revealed a substantial rate of lipid breakdown during this anabolic state (equivalent to approximately 25% of the newly synthesized triglyceride). We found equal rates of protein synthesis and breakdown in heart muscle (approximately 10% of the pool per day), consistent with the fact that the heart muscle mass did not change. In total, these findings demonstrate a novel application of the doubly labeled water method. Utilization of this approach, especially in unique rodent models, should facilitate studies aimed at quantifying the efficacy of interventions that modulate whole body carbon balance and lipid flux while in parallel determining their impact on (cardiac) muscle protein turnover. Last, the simplicity of administering doubly labeled water and collecting samples allows this method to be used in virtually any laboratory setting.
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PMID:Novel application of the "doubly labeled" water method: measuring CO2 production and the tissue-specific dynamics of lipid and protein in vivo. 1636 86

1. The testis of the ram secretes considerable amounts of amino acids (200mumoles/day) into the fluid collected from the efferent ducts. The principal amino acid in this testicular fluid is glutamate, which is present in concentrations about eight times those in testicular lymph or in blood from the internal spermatic vein. 2. The concentration of glutamate in seminal plasma from the tail of the epididymis is about ten times that in testicular fluid, and, though glutamate is the major amino acid in ejaculated seminal plasma, its concentration is less than in epididymal plasma. 3. After the intravenous infusion of [U-(14)C]glucose, labelled glutamate was found in the testicular fluid. Radioactivity was also detected in alanine, glycine, serine plus glutamine and aspartate. Alanine had the highest specific activity, about 50% of the specific activity of blood glucose. 4. When [U-(14)C]glutamate was infused, the specific activity of glutamate in testicular fluid was only about 2% that in the blood plasma. 5. Testicular and ejaculated ram spermatozoa oxidized both [U-(14)C]glutamate and [U-(14)C]leucine to a small extent, but neither substrate altered the respiration from endogenous levels. 6. No radioactivity was detected in testicular spermatozoal protein after incubation with [U-(14)C]glutamate or [U-(14)C]leucine. Small amounts of radioactivity were detected in protein from ejaculated ram spermatozoa after incubation with [U-(14)C]glutamate. 7. The carbon of [U-(14)C]glucose was incorporated into amino acids by testicular spermatozoa; most of the radioactivity occurred in glutamate.
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PMID:Amino acids in ram testicular fluid and semen and their metabolism by spermatozoa. 1674 31

Melanocortins are implicated in the control of energy intake/expenditure. Centrally administered melanotan II (MTII), a synthetic melanocortin 3/4-receptor agonist, decreases adiposity beyond that accountable by food intake decreases. Melanocortin-4 receptor (MC4-R) mRNA is expressed on sympathetic nervous system (SNS) outflow neurons to white adipose tissue (WAT) in Siberian hamsters, suggesting a role in lipid mobilization. Therefore, we tested whether third ventricular injections of MTII increased sympathetic drive to WAT and interscapular brown adipose tissue (IBAT) using norepinephrine turnover (NETO) as a measure of sympathetic drive. We also tested for MTII-induced changes in lipolysis-related WAT gene expression (beta3-adrenoceptors, hormone sensitive lipase) and IBAT thermogenesis (beta3-adrenoceptor, uncoupling protein-1). Finally, we tested whether third ventricularly injected MTII, a highly selective MC4-R agonist (cyclo[beta-Ala-His-D-Phe-Arg-Trp-Glu]NH2) increased or agouti-related protein decreased IBAT temperature in hamsters implanted with sc IBAT temperature transponders. Centrally administered MTII provoked differential sympathetic drives to WAT and IBAT (increased inguinal WAT, dorsosubcutaneous WAT and IBAT NETO, but not epididymal WAT and retroperitoneal WAT NETO). MTII also increased circulating concentrations of the lipolytic products free fatty acids and glycerol but not plasma catecholamines, suggesting lipid mobilization via WAT SNS innervation and not via adrenal medullary catecholamines. WAT or IBAT gene expression was largely unaffected by acute MTII treatment, but IBAT temperature was increased by MTII and the MC4-R agonist and decreased by agouti-related protein. Collectively, this is the first demonstration of central melanocortin agonist stimulation of WAT lipolysis through the SNS and confirms melanocortin-induced changes in BAT thermogenesis.
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PMID:Differential activation of the sympathetic innervation of adipose tissues by melanocortin receptor stimulation. 1770 43


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