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K D Roberts

Publications and source records attributed to K D Roberts.

At least 19 recordsLinked to original sources
PubMed1998

Synergistic effect of dexamethasone and isoproterenol on the expression of angiotensinogen in immortalized rat proximal tubular cells.

To investigate whether the expression of angiotensinogen (ANG) in rat kidney proximal tubules is stimulated by dexamethasone and isoproterenol, immortalized rat proximal tubular cells (IRPTC) were cultured in a monolayer. Immunoreactive rat ANG (IR-rANG) in the culture medium was measured by a specific radioimmunoassay (RIA) for rANG. This RIA was developed by employing rabbit antiserum against the purified recombinant rat ANG (rANG). The purified rANG from plasma and the iodinated rANG were used as the hormone standard and tracer, respectively. The RIA is specific for rat ANG and it has no cross-reactivity with other pituitary hormone preparations or other rat plasma proteins. The sensitivity of detection of the RIA is approximately 2 ng of rANG. The levels of IR-rANG in the culture media of IRPTC ranged from 2 to 5 ng/ml/24 hr/10(6) cells. The addition of dexamethasone (10(-13) to 10(-5) M) stimulated the expression and secretion of rANG from IRPTC in a dose-dependent manner, whereas the addition of isoproterenol alone had no effect. However, a combination of both dexamethasone and isoproterenol synergistically stimulated the expression and secretion of rANG by IRPTC. The synergistic effect of dexamethasone and isoproterenol was blocked by the presence of RU 486 (a glucocorticoid receptor antagonist) or propranolol (beta-adrenoceptor blocker). These studies suggest that the addition of dexamethasone and isoproterenol acts synergistically to stimulate the expression and secretion of ANG protein in rat proximal tubules in vivo.

Adrenergic beta-Agonists↗
PubMed1996

Selenium-vitamin E supplementation in infertile men. Effects on semen parameters and micronutrient levels and distribution.

In order to verify the hypothesis that selenium (Se) and vitamin E (Vit E) could improve male fertility, nine oligoasthenoteratozoospermic men were supplemented for a period of 6 mo with Se and Vit E. Compared to the baseline period (presupplementation) of 4 mo, statistically significant increases were observed for Se and Vit E levels, sperm motility, percent live, and percent normal spermatozoa. These improvements are likely to be "supplementation-dependent," since all of the parameters returned to baseline values during the posttreatment period. None of the couples reported a pregnancy during the study. The HPLC analysis conducted on the serum of one of the patients showed the existence of at least six different Se-containing peaks, whose Se content was affected by supplementation. The mechanism(s) involved in these improvements of semen parameters is presently under investigation.

Adult↗
PubMed1996

beta-Adrenoceptors and dexamethasone synergistically stimulate the expression of the angiotensinogen gene in opossum kidney cells.

We transiently co-transfected opossom kidney (OK) cells with the plasmid containing the cDNA for beta 1-adrenoceptor (pBC-beta 1 AR) or beta 2-adrenoceptor (pBC-beta 2 AR) and a fusion gene with the 5'-flanking region of the angiotensinogen (ANG) gene linked to a bacterial chloramphenicol acetyl transferase (CAT) coding sequence as a reporter, pOCAT (ANG N-1498/ +18). Co-transfection of plasmid pBC-beta 1 AR or pBC-beta 2 AR alone enhanced the expression of pOCAT (ANG N-1498/+18). The addition of isoproterenol further stimulated the expression of pOCAT (ANG N-1498/ +18) when co-transfected with pBC-beta 1AR, but not with pBC-beta 2AR. Moreover, the addition of a combination of dexamethasone and isoproterenol synergistically stimulated the expression of pOCAT (ANG N-1498/+18) when co-transfected with pBC-beta 1AR, but not when cotransfected with pBC-beta 2AR. The synergistic effect of dexamethasone and isoproterenol was inhibited by the presence of RU 486 (an antagonist of glucocorticoid) or Rp-cAMP (an inhibitor of cAMP-dependent protein kinase A I and II). To localize the putative cAMP-responsive element (CRE) and glucocorticoid responsive element (GRE) in the ANG gene, we constructed the fusion gene by inserting the DNA fragment, ANG N-806 to N-465 upstream of the thymidine kinase (TK) promoter fused to a CAT gene and introduced them with pBC-beta 1AR into OK cells. The addition of dexamethasone or isoproterenol alone stimulated the expression of pTKCAT (ANG N-806/-465). The addition of isoproterenol and dexamethasone synergistically stimulated the transcriptional activity of pTKCAT (N-806/-465). These studies demonstrate that the beta 1-adrenoceptor and dexamethasone act synergistically to stimulate the expression of the ANG gene in OK cells via the putative CRE and GREs in the 5'-flanking region of the rat ANG gene. These data should aid in the understanding of the molecular mechanism(s) of the stimulatory effect of catecholamines/glucocorticoid induced expression of the ANG gene in the kidney.

Adrenergic beta-Agonists↗
PubMed1995

Purification of lysophospholipase of human spermatozoa and its implication in the acrosome reaction.

The lysophospholipase of human spermatozoa was purified to homogeneity by sequential ion-exchange, gel filtration, and hydrophobic chromatography. The final preparation exhibited a single protein band on SDS-PAGE. The molecular mass of the enzyme was estimated to be 51 kDa by SDS-PAGE and 52 kDa by gel filtration. The optimal pH of this enzyme is 8.0. Polyclonal antibodies against lysophospholipase were prepared by placing the enzyme adsorbed on nitrocellulose directly into the spleen of rabbits. These antibodies were purified by protein A-agarose and by affigel-lysophospholipase chromatography. The purified antibodies and enzyme were used to study the possible role of lysophospholipase in the acrosome reaction. The addition of these antibodies led to an increase in the acrosome reaction, thus suggesting that inhibition of lysophospholipase produces a higher lysophosphatidylcholine concentration and results in an acrosome reaction level similar to that obtained by the calcium ionophore A23187. Immunofluorescence localization of the enzyme indicated that the enzyme is located on the head of spermatozoa. The purified sperm lysophospholipase and its specific antibodies represent important tools for the study of the regulation of this enzyme in reproductive processes. Furthermore, the study of this enzyme will allow evaluation of the mechanisms underlying the acrosome reaction.

Acrosome↗
PubMed1994

Major proteins of bovine seminal plasma inhibit phospholipase A2.

We have recently shown that the major proteins of bovine seminal plasma, namely BSP-A1, BSP-A2, BSP-A3 and BSP-30-kDa (collectively called BSP proteins) bind to spermatozoa and that the binding sites on the plasma membrane of spermatozoa are choline phospholipids. In view of the fact that these phospholipids are substrates for phospholipase A2 (PLA2), a key enzyme in sperm capacitation and the acrosome reaction, the effect of BSP proteins on this enzyme activity was investigated. Since these BSP proteins are ubiquitous, the effect on pig pancreatic PLA2 was also studied. In contrast with control proteins, when preincubated with phosphatidylcholine as substrate, all BSP proteins inhibited both pancreatic and sperm PLA2 activity in a dose-dependent manner and in the presence of 1-6 microM BSP protein the enzyme activity was completely abolished. When phosphatidylethanolamine was used as substrate, only pancreatic PLA2 was inhibited. On the other hand, when the BSP proteins were preincubated with the enzyme followed by addition of substrate, a biphasic effect was observed; there was stimulation of enzyme activity below 1.3 microM BSP followed by an inhibition above this concentration. The inhibitory activity was trypsin-sensitive but heat-resistant. The effect of co-incubation of heparin, which is implicated in sperm capacitation and which also interacts with BSP proteins, was studied. Heparin (10 microM) had no effect on the PLA2 inhibitory activity exhibited by all BSP proteins. The PLA2 inhibitory effect exhibited by BSP proteins was abolished with excess substrate. The BSP proteins were adsorbed on PLA2-agarose and could be affinity cross-linked to the enzyme, indicating a direct interaction of enzyme with the inhibitor. These results suggest that these BSP proteins modulate PLA2 activity and therefore, phospholipid metabolism.

Animals↗
PubMed1994

Flow cytometric evaluation of the acrosome reaction of human spermatozoa: a new method using a photoactivated supravital stain.

A flow cytometric assay using a double-stain method for the measurement of the acrosome reaction of human spermatozoa is described. The use of a stable photoactivated stain, ethidium monoazide, allowed evaluation of the viability of spermatozoa. This stain was more stable in fixed samples than propidium iodide, which is not bound covalently to DNA and is therefore removed readily during the washing procedure. The permeabilized acrosome was labelled with Pisum sativum agglutinin conjugated with fluoroisothiocyanate. Since this lectin binds to the acrosome and acrosomal contents, a decrease in the fluorescence intensity allows the cytometric evaluation of the acrosome reaction. Microscopic analysis and flow cytometric analysis were well correlated and cell sorting was performed to ensure the homogeneity of each different subpopulation encountered.

Acrosome↗
PubMed1993

Effect of platelet-activating factor, lyso-platelet-activating factor, and lysophosphatidylcholine on sperm motion: importance of albumin for motility stimulation.

OBJECTIVES: To determine the effect of platelet-activating factor (PAF), the PAF derivative lyso-PAF, and lysophosphatidylcholine on in vitro sperm motility and to determine the role of albumin in this interaction. DESIGN: Washed human spermatozoa were exposed to a range of PAF, lyso-PAF, or lysophosphatidylcholine concentrations, supplemented with different albumin concentrations, and the effect on sperm motion was quantified with a computer-assisted motion analysis. The metabolism of these compounds by spermatozoa was also assessed. SETTING: University research laboratory. PATIENTS, PARTICIPANTS: Semen samples were obtained from donors and patients attending an infertility clinic. INTERVENTIONS: Human spermatozoa were incubated with PAF, lyso-PAF, or lysophosphatidylcholine at 10(-11) to 6 x 10(-4) M, with 0% to 1.2% albumin, and motility was evaluated at different time periods from 5 to 240 minutes. Tritiated PAF, lyso-PAF, or lysophosphatidylcholine was incubated with spermatozoa, and the metabolites were separated and quantified by thin-layer chromatography (TLC). MAIN OUTCOME MEASURES: Sperm motion characteristics, including the percentage of motile spermatozoa, velocity, and linearity, and sperm viability were determined. The metabolism of PAF, lyso-PAF, and lysophosphatidylcholine by spermatozoa was also studied. RESULTS: Fifty micromolar of PAF and 100 microM lyso-PAF, supplemented with 0.3% albumin, increased sperm linear velocity by 41% +/- 5% (+/- SEM) and 44% +/- 5% and curvilinear velocity by 17% +/- 3% and 21 +/- 3%, respectively. Lysophosphatidylcholine had a similar effect but only at 22 degrees C and not 37 degrees C. In the absence of albumin, neither PAF, lyso-PAF, or lysophosphatidylcholine induced increases in sperm motion. Lysophosphatidylcholine and lyso-PAF are not detectably metabolized by spermatozoa, whereas 12.5% +/- 1.2% of PAF is hydrolyzed to lyso-PAF in 1 hour. CONCLUSION: Platelet-activating factor, lyso-PAF, and lysophosphatidylcholine independently stimulate sperm linear and curvilinear velocity. This action requires albumin. These compounds may be of use in the treatment of asthenozoospermic males.

Fertilization↗
PubMed1993

Platelet-activating factor acetylhydrolase in the male reproductive tract: origin and properties.

The properties and origin of platelet-activating factor acetylhydrolase- (PAF-AH)-like activity in the male reproductive tract were investigated. Seminal plasma (SP) and serum were obtained from normal donors and infertile patients while prostatic fluid (PF), seminal vesicle fluid (SVF) and vas deferens fluid were collected at autopsy. PAF-AH-like activity was found in all fluids tested. The specific activity of the enzyme in SP and PF was twice that of PAF-AH in serum and 15-fold higher than that in SVF and vas deferens fluid. In SP, PAF-AH-like activity was Ca(++)-independent, acid and heat labile, stable to freezing, not inhibited by phosphatidylcholine, but was inhibited by 10 mM disopropylfluorophosphate (DFP) and 13 mM phenylmethylsulfonylfluoride (PMSF). These data indicate that the properties of the enzyme in SP are similar to those reported for PAF-AH in serum. The variation in specific activity of PAF-AH in reproductive tract fluids suggest that there are either activators of PAF-AH in SP or inhibitors in one or several of the other reproductive tract fluids.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1993

Calmodulin-binding proteins in bovine semen.

An 125I-labelled calmodulin gel overlay procedure was used to direct calmodulin-binding proteins in bovine spermatozoa and seminal plasma. Several calmodulin-binding proteins with molecular masses ranging from 12 to > 200 kDa were detected in epididymal and ejaculated spermatozoa. Certain of these proteins exhibited preferential calmodulin-binding in the presence of Ca2+, while others exhibited binding only in its absence. In seminal plasma, only two major proteins with molecular masses of 15 and 16 kDa showed a higher calmodulin-binding activity in the presence of Ca2+, whereas several polypeptides in the range of 6-17 kDa bound higher amounts of radiolabelled calmodulin in the absence of Ca2+. Our previous study has shown that a group of closely related major proteins, designated as BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa, isolated from bovine seminal plasma (BSP) have molecular masses in the range of 15-30 kDa. This prompted us to investigate whether these polypeptides from bovine seminal fluid interact with calmodulin. The results indicated that calmodulin binds to purified BSP-A1, -A2, -A3 and BSP-30 kDa proteins in the presence and absence of Ca2+. Furthermore, many polypeptides of low molecular mass (6-14 kDa) in bovine seminal plasma that crossreact with these BSP proteins also show high calmodulin-binding activity, particularly in the absence of calcium. This was further demonstrated following the limited proteolysis of the BSP proteins. Several tryptic-peptides of BSP-A1/-A2 and BSP-30 kDa exhibited higher calmodulin-binding activity than the intact BSP proteins. In view of the key role of Ca2+ in triggering the acrosome reaction and the role of calmodulin in intracellular transport of calcium, it is suggested that BSP proteins are involved in sperm capacitation and the acrosome reaction.

Animals↗
PubMed1993

Partial characterization of galactosyltransferase in human seminal plasma and its distribution in the human epididymis.

Galactosyltransferase activity has been partially characterized in human seminal plasma. Km values of 130 mumol l-1 for UDP-galactose and 2.25 mmol l-1 for N-acetylglucosamine were calculated and the enzyme was found to be dependent on temperature and manganese and present as a highly active component of human seminal plasma. Galactosyltransferase was inhibited by nucleotides, glycosylated nucleotides, bovine and human alpha-lactalbumin but not by monosaccharides. Radiation inactivation studies revealed that the biologically active unit of seminal plasma galactosyltransferase has a molecular mass of 45 kDa. Although the majority of galactosyltransferase activity found in seminal plasma is probably of prostatic origin, we report for the first time that it is also present in human epididymal intraluminal fluid. Low activity was detected in the proximal caput region but activity increased to maximum values in the adjacent downstream segment, the intermediate caput region. Specific activity was relatively constant albeit at a lower value in the following epididymal segments and vas deferens. The significance of the epididymal and seminal plasma galactosyltransferase activities is unknown, but the enzyme could be implicated in glycosylation events that are known to be important in gamete interaction.

Animals↗
PubMed1993

Distribution of lysophospholipids and metabolism of platelet-activating factor in human follicular and peritoneal fluids.

The distribution of lysophosphatidylcholine, lyso-platelet-activating factor and platelet-activating factor (PAF) was studied in human plasma and in follicular and peritoneal fluid. In plasma, peritoneal and follicular fluids, 51%, 87% and 89%, respectively, of the total lipids were found in the protein fraction (the density > 1.21 fraction). Two forms of lysophospholipids were identified in this fraction: one of high affinity and one of low affinity for albumin. The metabolism of PAF in human follicular fluid, peritoneal fluid and plasma was also investigated. PAF-acetylhydrolase activity was found in both peritoneal and follicular fluids which induced a time-dependent hydrolysis of [3H]PAF. The half-life of PAF was estimated to be 7-12 min in plasma, 15-25 min in peritoneal fluid and approximately 2 h in follicular fluid. PAF-acetylhydrolase activity in embryo culture media supplemented with 10% serum was markedly inhibited by addition of commercial serum albumin. When 25 g albumin l-1 was added, 22% of [3H]PAF was hydrolysed h-1 compared with 72% in media without albumin. The concentrations of lysophosphatidylcholine measured in plasma, in follicular and peritoneal fluids were 252, 286 and 53 mumol l-1, respectively. The distribution of these lysophospholipids and the metabolism of PAF in the female genital tract fluids reported in the present study provide evidence for the involvement of these biologically active lipid mediators in a variety of reproductive processes including sperm-egg interactions and embryonic development.

Albumins↗
PubMed1993

Interference of lysophosphatidylcholine in hormone radioimmunoassays.

The interference of synthetic and naturally occurring detergents in immunoassays is well documented. In the present study, we evaluated the effect of lysophosphatidylcholine (LPC) and found that the lysophospholipid interfered with formation of the antigen-antibody complex in hormone immunoassays. In the presence of LPC (100 mumol/L), progesterone was overestimated by 29%. Furthermore, physiological concentrations of LPC (140 mumol/L) interfered with the assays of cortisol, progesterone, and aldosterone, resulting in overestimations of 35%, 30%, and 27%, respectively. The addition of albumin decreased the interference by LPC to 7% in the assay of cortisol and progesterone when the LPC:albumin ratio was unity. Adding cholesterol (100 mumol/L) also reduced by 50% the interference induced by LPC. Finally, treating plasma to increase the endogenous LPC concentration also resulted in interference in the cortisol assay. Thus, interpretation of the results of these assays should take into consideration the endogenous serum albumin:LPC ratio.

Aldosterone↗
PubMed1992

The zona pellucida binds the mature form of an oviductal glycoprotein (oviductin).

The use of a monoclonal antibody (MAb) specific for the oviductal zona pellucida (ZP) of the hamster has demonstrated that a new antigen (oviductin) is acquired by the ZP during transit of the oocyte in the oviduct. The epitope that is recognized by the MAb bears a terminal N-acetyl-D-galactosamine residue. We conducted a study in order to determine whether this immunoreactivity of the oviductal ZP results from the addition of the terminal sugar residue to a preformed ZP protein or from the transfer of the mature glycoprotein produced by oviductal secretory cells. We measured the incorporation of [35S]methionine into proteins using four different incubation systems: cumulus oophorus (CO) alone, CO in the presence of oviductal fluid, CO co-incubated with empty oviducts, and CO within intact oviducts. At the end of the incubation period, the ZP, vitelli, dispersed cumulus without oocyte, oviducts, and culture medium were isolated and analyzed for their protein content by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), autoradiography, and immunodetection. The cumulus cells synthesized several proteins, independently of the oviductal environment; however, none of these proteins corresponded to oviductin. The ZP and the vitelli of cumulus oophorus that were incubated either alone or in the presence of oviductal fluid did not contain radioactive oviductin. When the oviduct (empty or intact) was present in the incubation system, radiolabeled oviductin was synthesized and secreted into the incubation medium. The ZP picked up a detectable amount of radioactive antigen only in the system in which intact oviducts were incubated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The phospholipase A2 of human spermatozoa; purification and partial sequence.

In view of its proposed key role in the acrosome reaction, phospholipase A2 has been isolated and purified from human spermatozoa. Following SDS-PAGE, a single major band was obtained with an estimated molecular mass of 16.7 kDa. Sequence analysis of the N-terminal portion of the molecule revealed the identity of the first 19 amino acids to be YNYQFGLMIVITKGHFAMV. From this partial analysis it is evident that the phospholipase A2 of human spermatozoa represents a new sequence. Of interest is the location of glutamine-4, phenylalanine-5, methionine-8 and isoleucine-9; this sequence appears to be highly conserved throughout evolution.

Amino Acid Sequence↗
PubMed1992

Platelet-activating factor acetylhydrolase in human seminal plasma.

OBJECTIVE: To search for the presence of platelet-activating factor (PAF) acetylhydrolase activity in human seminal plasma. DESIGN: Experimental. SETTING: Reproductive laboratory in a university-affiliated hospital research center. PARTICIPANTS: Human male volunteers were selected on the basis of apparent normal health. RESULTS: Human seminal plasma contained significant levels of PAF-acetylhydrolase activity. Enzymatic hydrolysis of PAF displayed typical kinetics and was a calcium-independent process. Platelet-activating factor acetylhydrolase in seminal plasma was associated with a very high-density lipoprotein fraction. Enzymatic activity was independent of sperm count and motility. CONCLUSION: Human seminal plasma contains significant levels of PAF acetylhydrolase. This enzyme may be an important factor in the regulation of PAF concentration and activity.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1991

Prostatic epithelial cells in culture: phosphorylation of protein tyrosyl residues and tyrosine protein kinase activity.

The ability of dividing canine prostatic epithelial cells in primary monolayers to phosphorylate protein tyrosyl residues was evaluated by metabolic studies performed through incorporation of [32P]-phosphate into alkali-resistant phosphoproteins and by the assay of their tyrosine protein kinase activity. The presence of sodium orthovanadate during cell incubation with [32P]-phosphate greatly enhanced the relative labelling intensity of a 44 kDa alkali-resistant phosphoprotein and the total cellular content of phosphotyrosine in proteins; in this respect, growth factors such as epidermal growth factor, insulin, and insulin-like growth factor I, and the steroids dihydrotestosterone and estradiol were inactive. When the cells were solubilized, sodium orthovanadate stimulated their tyrosine protein kinase activity and inhibited their phosphotyrosine phosphatase activity. To characterize the tyrosine protein kinase of these cultured cells, conditions for optimal activity were established using the substrate poly [Glu80Na, Tyr20]. The subcellular localization of the enzyme was determined upon cell fractionation: 88% of the kinase activity was associated with the particulate fraction and 30% of this activity was partially solubilized with 0.5% Triton X-100; this solubilization was improved to 83% in the presence of 0.25 M KCI. The enzyme directly solubilized from prostatic cells with Triton X-100 (38% of activity) mainly catalyzed the alkali-resistant phosphorylation of pp63, pp59, and pp44, which contained phosphotyrosine. These proteins were also phosphorylated by the major peak of kinase activity which was eluted at an apparent molecular weight of 300-350 kDa upon gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies.

The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. Immunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bands of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.

Adult↗