Platelet activating factor and pentoxifylline as human sperm cryoprotectants.

OBJECTIVE: To explore whether the addition of platelet-activating factor (PAF) or pentoxifylline before cryopreservation improves the recovery of motile viable sperm and what role cyclic adenosine monophosphate (cAMP) plays in this recovery. DESIGN: Washed sperm was cryopreserved in the absence of and in the presence of PAF and pentoxifylline. After 2 weeks these samples were quick-thawed and evaluated before and after washing for sperm motility and other motion characteristics. Sperm viability and cAMP concentration were determined to compare the effects of these cryoprotectants. RESULTS: When sperm samples were cryopreserved in the presence of PAF or pentoxifylline, an improvement in the recovery of motile sperm in unwashed and washed post-thaw samples was observed. There were 38% more motile sperm recovered with PAF and 15% more with pentoxifylline when compared with untreated samples. In comparison with the unwashed samples, sperm motility in post-thaw samples was lowered by the washing procedure. When PAF was used as a cryoprotectant, a significant improvement in the linearity and straight line velocity of the post-thaw sperm was observed. When pentoxifylline was used as a cryoprotectant, lateral head displacement was significantly improved in the post-thaw samples than in the control group. Both PAF-and pentoxifylline-treated samples contained a greater number of viable sperm than the control. The cAMP concentrations in post-thaw samples were 12-fold higher in pentoxifylline-treated samples and 4-fold higher in PAF-treated samples when compared with the untreated control. A 4-fold decrease in cAMP concentration was observed in post-thaw control samples compared with fresh-washed sperm. CONCLUSIONS: The results of this study suggest that both PAF and pentoxifylline are useful cryoprotectants for the increased recovery of motile, viable sperm. Although increased recovery of motile sperm in pentoxifylline-treated samples is related to higher cAMP levels, the cryoprotective effect of PAF does not appear to be due to increased cAMP.

Characterization of a trypsin inhibitor from equine urine.

A trypsin inhibitor was isolated from pregnant mares' urine by adsorption on bentonite and elution with aqueous pyridine followed by batch DEAE-cellulose treatment and column chromatography. Final purification to an electrophoretically homogenous glycoprotein was achieved by gel permeation chromatography. This equine urinary trypsin inhibitor (E-UTI) is acid- and heat-stable, has a molecular weight of 22 to 23 kDa, an isoelectric point of 4.55, forms a 1:1 molar complex with trypsin and has serine as its N-terminal amino acid. The N-terminal amino acid sequence of this protein is almost identical with that of EI-14, the inhibitor obtained from horse serum by tryptic treatment, except for two extra amino acid residues, Ser-Lys- on the N-terminal end of E-UTI. In its isoelectric point E-UTI differs from EI-14 and the inhibitor from human urine.

Growth hormone-releasing peptide (GHRP) binding to porcine anterior pituitary and hypothalamic membranes.

The synthetic hexapeptide, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP, Growth Hormone-Releasing Peptide), has no structural similarities with any of the GH-releasing peptides known and its action in releasing GH is by a complementary but yet not clearly defined action on the pituitary as well as hypothalamus. Therefore, in vitro studies have been performed to demonstrate and characterize GHRP binding sites on peripheral membranes of both porcine pituitary and hypothalamus. The membrane binding sites were specific, reversible, saturable and time, temperature, pH and concentration dependent under optimum binding assay conditions. The maximum specific binding was observed between pH 5.0 and 6.0. In the presence of Ca2+ and Mg2+ ions, with or without chelating agents there was a significant reduction in the specific binding. Scatchard analysis of these binding sites using increasing doses of unlabeled GHRP revealed a single low affinity site with a 2.1 x 10(-5) M and 1.7 x 10(-5) M and a maximum number of sites of 10 nmol/mg protein and 5 nmol/mg protein for pituitary and hypothalamus, respectively. It is also observed that (D-Lys3)-GHRP, substance P antagonists and growth hormone-releasing factor analog were potent inhibitors of GHRP binding in both tissues.

Demonstration and characterization of the specific binding of growth hormone-releasing peptide to rat anterior pituitary and hypothalamic membranes.

Since the growth hormone-releasing peptide (GHRP), His-D-Trp-Ala-Trp-D-Phe-Lys-NH2, was found to specifically release growth hormone by a complementary but yet not clearly defined action on the pituitary as well as the hypothalamus, in vitro studies have been performed to demonstrate and characterized GHRP binding sites on peripheral membranes of both the rat anterior pituitary and hypothalamus. Optimum binding assay conditions were established using [125I]Tyr-Ala-GHRP as the radioligand. The membrane binding sites were specific, reversible, saturable and time, temperature, pH and concentration dependent. Computerized analyses of competition experiments suggested two classes of binding sites in both pituitary and hypothalamic membranes. The maximum specific binding was observed at pH 5.0 than the physiological pH in both tissues. Pretreatment of the membranes with trypsin prevented specific binding. The increase in Bmax was statistically significant and showed a 2.0- to 8.9-fold and 5.8- to 11.2-fold in pituitary and hypothalamus, respectively, whereas the affinity constants (Kds) were not significant. Of the synthetic and natural neuropeptides that influence the release of GH from somatotrophs, only (D-Lys3)GHRP, substance P antagonists and growth hormone-releasing factor analog were potent inhibitors of GHRP binding in both tissues.

Purification and characterization of two distinct lipases from Geotrichum candidum.

Lipase, an enzyme that hydrolyzes triacylglycerol, has been purified and characterized. The purification procedure includes ethanol precipitation and chromatographies on Sephacryl-200 HR, high resolution anion-exchange (mono Q) and Polybuffer exchanger 94. With this procedure, two forms of lipases from Geotrichum candidum were obtained. Lipase I (main enzyme) and lipase II (minor enzyme) were purified 35-fold with a 62% recovery in activity and 94-fold with a 18% recovery in activity, respectively. Their molecular weights have been estimated by polyacrylamide gel electrophoresis under denaturing conditions and by molecular sieving under native conditions at 56,000. Lipase I and II had optimum pH values of 6.0 and 6.8 and isoelectric points of 4.56 and 4.46, respectively. The enzymes are stable at a pH range of 6.0 to 8.0. Monovalent ions had little effect on both enzyme activities, while divalent ions at concentrations above 50 mM inhibited the lipase activities in a concentration-dependent manner. Sodium dodecyl sulfate at a concentration lower than 10 mM completely inhibited the lipase activity.

A simple and sensitive method for the estimation of microbial lipase activity.

A simple and sensitive method for the estimation of microbial lipase activity is described. In this method, lipase (EC 3.1.1.3) is incubated with an emulsified substrate and the fatty acid formed is estimated by high-performance liquid chromatography. The emulsified substrate, triolein, is prepared with various solubilizers, bovine serum albumin, gelatin, ovalbumin, gum arabic, Triton X-100, and n-octyl-glucopyranoside, in suitable buffers. The oleic acid, one of the products formed in the reaction, is separated on an ODS column with a premixed acetone-acetonitrile solvent system. Results with various lipase activities showed gum arabic to be the best among various solubilizers used to prepare the emulsified substrate. Also the effects of mono- and divalent ions on microbial lipase activities are analyzed. Finally, this method is compared with two other methods, titration and reversed micelles, and found to be simple and more sensitive.

Studies on two major contaminating proteins of the cytoplasmic inclusion bodies in Escherichia coli.

Two major contaminating proteins of the cytoplasmic inclusion bodies in recombinant Escherichia coli overexpressing a LacZ-papain fusion protein were studied. The proteins were isolated and their amino acid composition and N-terminal sequence analyses were performed. The N-terminal sequence of these proteins were then compared with the sequence of E. coli proteins available in the data bank. The sequences of the contaminating proteins matched with the outer membrane protein II and outer membrane protein F of E. coli.

Mammalian protein methylesterase. Physical and enzymic properties.

Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.

Stoichiometric carboxyl methylation of chromogranins from bovine adrenal medullary cells.

The secretory proteins from adrenal chromaffin granules, chromogranins A, A1 and A2, were found to be excellent in vitro methyl acceptor proteins. The purified protein-carboxyl methylase (PCM) from bovine erythrocytes incorporated 660, 2540 and 7890 pmol of methyl group/10 min/mg protein in chromogranins A, A1 and A2, respectively. However, dopamine beta-hydroxylase, another secretory protein within chromaffin granules was poorly methylated. The stoichiometry of methylation for chromogranins A, A1 and A2 was 0.26, 0.89 and 1.3 mol of methyl group/mol of protein, respectively. Kinetic analysis showed that chromogranins A1 had the highest turnover rate followed by chromogranins A2 and A. These chromogranins are the first secretory proteins to be stoichiometrically methylated in vitro without prior deamidation.

Protein-carboxyl methylation in adrenal medullary cells.

1. The protein-carboxyl methylating system has been studied in adrenal medullary cells either using disrupted cell components or with intact cells. Whereas the enzyme protein-carboxyl methylase (PCM) is cytosolic, the majority of its substrates is on or within chromaffin granules. With intact granules, methylation of surface proteins results in solubilization of membrane proteins. 2. Membrane PCM substrates have been identified as two proteins with apparent molecular weights of 55,000 and 32,000. Among the substrates located inside the granules, the chromogranins are excellent substrates, while dopamine beta-hydroxylase is poorly methylated. 3. Under physiological conditions, stimulation of the splanchnic nerve results in an increase in adrenal medullary protein-methyl ester formation as well as in an augmented methanol production. With adrenal medullary cells in culture, carboxyl-methylated chromogranin A is detected in mature chromaffin granules between 3 and 6 hr after labeling. Methylated chromogranins are secreted concomitantly with catecholamines following cholinergic stimulation. 4. These data coupled with those of Chelsky et al. (J. Biol. Chem. 262:4303-4309, 1987) on lamin B suggest that PCM methylates residues other than D-aspartyl and L-isoaspartyl in proteins. They further suggest that methylation may occur on nascent peptide chains before they are injected into the rough endoplasmic reticulum.

Leupeptin and chymostatin inhibit mammalian protein methylesterase activity.

Purified protein methylesterase (PME) from rat kidneys was incubated with ovalbumin-methyl esters and a series of protease inhibitors. All four inhibitors with C-terminal aldehyde, leupeptin, chymostatin, Boc-Gln-Leu-Lys-H and D-Phe-Pro-Arg-H completely blocked PME activity. Other inhibitors including, alpha-1 antitrypsin, soybean trypsin inhibitor, antithrombin III, phenylmethylsulfonylfluoride, aprotinin and lima bean trypsin inhibitor had no significant effect whereas pepstatin, at high concentration reduced the enzymatic activity by 25%. The most potent inhibitors, leupeptin and chymostatin, had a Ki of 3.5 X 10(-8) and 5.4 X 10(-7) M, respectively. These inhibitors provide two new tools to study PME function.

Antagonism of acute feeding response to 2-deoxyglucose and 5-thioglucose by GABA antagonists: the relative role of ventromedial and lateral hypothalamus.

The effect of GABA antagonists picrotoxin and bicuculline was studied on hyperphagia caused by 2-DG and 5-TG. The GABA antagonists were administered either SC or into the VMH or LH through stereotaxically implanted chronic cannulae. The peripheral as well as VMH injection antagonised the hyperphagia significantly. In contrast, injection of these agents into the LH failed to produce any effect. These findings show that in a glucoprivic state there might be an increased GABAergic activity in the VMH.

Effect of 5-thio-D-glucose on testosterone biosynthesis in vivo in mice testes.

Testicular synthesis of (14C)cholesterol and (14C)testosterone from (14C)acetate were investigated in mice treated with 5-thio-D-glucose at a dose of 33 mg/kg body weight/day for 21 days. The testicular synthesis of free cholesterol as well as steroids were significantly decreased. The steroid synthesizing enzymes, cholesterol esterase, cholesterol side-chain cleaving enzyme, total alpha-hydroxysteroid dehydrogenase and total beta-hydroxysteroid dehydrogenase, were also analysed. Cholesterol esterase and total beta-hydroxysteroid dehydrogenase were significantly reduced whereas total alpha-hydroxysteroid dehydrogenase was unaffected. Hence, a decrease in free cholesterol for steroid synthesis and a decreased activity of the steroidogenic enzyme, beta-hydroxysteroid dehydrogenase, were responsible for the diminished synthesis of testosterone.

Glucose concentration and insulin release in 5-thio-D-glucose-treated mice.

Male albino mice were given a single dose of various concentrations (25, 50, and 100 mg/kg body weight) of 5-thio-D-glucose or daily infusions (33 mg/kg body weight) of 5-thio-D-glucose for 21 days. Elevated blood glucose and immunoreactive insulin (IRI) levels were observed in the mice treated with 5-thio-D-glucose. Fasting glucose levels reached a maximum in 30 minutes and IRI levels reached a maximum in 60 to 90 minutes in the single-dose treated animals compared to preintubation levels. In the mice treated for 21 days, the fasting and fed glucose and IRI levels were significantly increased. Single dose of glucose (1 g/kg body weight) given to fasting and fed mice did not alter the glucose and IRI levels in the treated animals. However, a single dose of 5-thio-D-glucose (33 mg/kg body weight) given to fasting and fed treated animals increased the IRI levels significantly but not the glucose concentration. These data show that both single-dose and 3-week treatment with 5-thio-D-glucose produced a hyperinsulinemic diabetes in male albino mice.

Effect of 5-thio-D-glucose on testicular lipids of mice.

Spermatogenesis is reported to be completely inhibited by 5-thio-D-glucose in mice. In an investigation of this inhibition, testicular lipid constituents, namely, total lipids, phospholipids, triacylglycerol, free and total cholesterol, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and NADPH generators like glucose-6-phosphate dehydrogenase, isocitrate dehyrogenase and malic enzyme were estimated in mice fed with 5-thio-D-glucose (33 mg/kg) by gastric intubation for 21 days. Significant increase in cholesteryl ester, glucose-6-phosphate dehydrogenase activity and malic enzyme and a decrease in free cholesterol and phospholipids were observed.