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Biomedical subjects

J G Chafouleas

Publications and source records attributed to J G Chafouleas.

10 recordsLinked to original sources
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

Decrease in calmodulin concentrations during heparin-induced capacitation in bovine spermatozoa.

Concentrations of the intracellular Ca(2+)-mediator calmodulin (CaM), were measured by radioimmunoassay during heparin-induced capacitation of bull spermatozoa. Heparin reduced sperm CaM concentrations in a dose-dependent manner corresponding with an increase in in-vitro fertilization rates. Such reductions were observed after heparin treatment for 4-6 h, which is in agreement with the length of the capacitation period in bulls and was concomitant with an increase in CaM concentration in the incubation medium, suggesting translocation of CaM from the spermatozoa to the surrounding milieu. This CaM translocation was inhibited partly by the protease inhibitor benzamidine, suggesting a role for the sperm protease in this process.

Acrosome↗
PubMed1991

Activation of bovine rod outer segment phosphatidylinositol-4,5-bisphosphate phospholipase C by calmodulin antagonists does not depend on calmodulin.

Calmodulin antagonists stimulated phosphatidylinositol-4,5-bisphosphate phospholipase C in soluble and particulate fractions of bovine rod outer segments. Antagonists tested include trifluoperazine, melittin, calmidazolium, compound 48/80, W-13 [N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide], and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. All were effective, but W-7 was chosen for further characterization of the effect, which was most pronounced in the soluble fraction. Phospholipase C activity in the soluble fraction did not increase linearly with the quality of enzyme assayed, suggesting the presence of an endogenous inhibitor or an inhibitory self-association of the enzyme. W-7 appeared to counteract this inhibition, resulting in a linear activity-quantity relationship. Stimulation by W-7 was therefore largest when large amounts of crude enzyme were assayed and small or nil when small amounts were assayed. The effect of W-7 was also dependent on [Ca2+], with half-maximal stimulation occurring between 0.1 and 1 microM. W-7 and W-13 were much more effective than their nonchlorinated analogues W-5 and W-12 at increasing phospholipase C activity. While this pattern of effectiveness is typical of calmodulin-mediated processes, the absence of any effect by added calmodulin and the retention of W-7 sensitivity by purified CaM-free enzyme argue against regulation by CaM. Octyl glucoside, a nonionic detergent, mimicked some of the effects of CaM antagonists, suggesting that the antagonists act by interfering with protein-protein interactions. It appears likely that CaM antagonists prevent an inhibitory multimerization or aggregation of at least one form of ROS phospholipase C.

Animals↗
PubMed1990

Increased sensitivity to Vinca alkaloids in cells overexpressing calmodulin by gene transfection.

Mouse C127 cells, transfected with the chicken calmodulin (CaM) gene and overexpressing CaM protein, were used to evaluate the effect of elevated levels of CaM on the sensitivity of these cells to various anticancer drugs. Clones C2 and C3 overexpress CaM mRNA by 40- and 80-fold, respectively, and CaM protein 3- and 8-fold, respectively. These cell lines were tested for their sensitivity to vincristine, vinblastine, bleomycin, and Adriamycin by comparing the 50% inhibitory concentration in a 72-h growth inhibition assay. The 50% inhibitory concentration values for vincristine with C2 and C3 cells were 6.27 +/- 0.56 nM and 6.60 +/- 0.96 nM, respectively. These values were significantly lower than 13.9 +/- 0.79 nM for the parental C127 cells and 14.0 +/- 1.55 nM for clone 6.8 (the control cell line for transfection without the chicken CaM gene) at P less than or equal to 0.005. The proliferation of C2 and C3 cells was inhibited at lower concentrations of vinblastine as well. The 50% inhibitory concentration values for the C2 and C3 cell lines were approximately one-half those required for C127 or clone 6.8 cells. However, no significant difference in the sensitivity to the DNA-binding drugs, bleomycin and Adriamycin, was observed between the different cell lines. The uptake of [3H]vinblastine was evaluated and found to be increased 1.6- and 2.8-fold in C2 and C3 cells, respectively, as compared with that value obtained for C127 cells. Moreover, the efflux of [3H]vinblastine from vinblastine-loaded cells was also observed to be decreased in the C2 and C3 cell lines. These data suggest that the increase in CaM expression in the C2 and C3 cell lines might be related to the higher sensitivity of these cells to Vinca alkaloids. This increased sensitivity appears to be due to the increase in intracellular concentration of the Vinca alkaloids as a result of an increase in drug uptake and a decrease in efflux. Moreover, the increased sensitivity of clones C2 and C3 to Vinca alkaloids appears to be specific to this class of drugs in that no collateral effects were observed for the DNA-damaging drugs, Adriamycin and bleomycin.

Animals↗
PubMed1990

Decreased binding of calmodulin to bull sperm proteins during heparin-induced capacitation.

The 125I-calmodulin gel overlay procedure was used to evaluate the effect of a heparin treatment on the calmodulin-binding proteins of bull spermatozoa. At concentrations that increase the in vitro fertilization rate of in vitro-matured oocytes, heparin induced a decrease in the binding to calmodulin (CaM) in 3 sperm proteins of 28, 30, and 49 kDa. The binding of these proteins to CaM was higher when Ca2+ was absent from the overlay procedure, and this binding was negatively correlated to the fertilization rate. These results suggest that sperm capacitation is associated with a decrease in the binding of CaM to the 28, 30, and 49 kDa sperm CaM-binding proteins. Implications of such a decrease are discussed.

Animals↗
PubMed1990

Identification of a 51-kilodalton calmodulin binding protein that changes during estrogen-stimulated cell growth.

Calmodulin-binding proteins (CaMBPs) were analyzed during estrogen-stimulated growth in the human breast cancer cell line ZR-75-1. A variety of Ca2(+)-dependent and -independent CaMBPs were observed to be present in these cells. Calmodulin (CaM) binding to a 51-kilodalton protein was shown to be Ca2(+)-dependent. Moreover, binding to this protein was reduced in the estrogen-treated cells. This effect occurred early during estrogen-stimulated cell growth and was maintained during exponential growth in the presence of estrogen. 125I-labeled CaM overlay procedure of two-dimensional polyacrylamide gels reveals that this 51-kilodalton protein is composed of at least two distinct isoforms with different isoelectric points. Subcellular localization demonstrates that this protein resides exclusively in the microsomal fraction.

Breast Neoplasms↗
PubMed1989

Effect of heparin on the expression of calmodulin-binding proteins in bull spermatozoa.

A 125I-labelled calmodulin gel overlay procedure in the presence and the absence of Ca2+ was used to evaluate bull spermatozoa calmodulin-binding proteins. Frozen spermatozoa were thawed, washed and incubated for 6 h before being processed for SDS polyacrylamide gel electrophoresis and the 125I-labelled calmodulin gel overlay procedure. In non-incubated spermatozoa, up to 14 binding proteins were detected. Some exhibited greater calmodulin binding in the presence of Ca2+ while others exhibited greater binding when Ca2+ was absent. When heparin (2 micrograms/ml) was present in the incubation medium, a decrease in the calmodulin binding to the proteins of Mr 28,000 and 30,000 was detected in the presence of Ca2+ and EGTA. This effect of heparin was time- and dose-dependent and was increased by the presence of the acrosin inhibitor benzamidine. Sperm capacitation could thus be related to a decrease in the binding of calmodulin to these proteins.

Animals↗
PubMed1988

Developmental appearance of the Ca2+-binding proteins parvalbumin, calbindin D-28K, S-100 proteins and calmodulin during testicular development in the rat.

Calcium and intracellular Ca2+-binding proteins are possibly involved in hormone production and spermatogenesis in rat testis. Parvalbumin, calbindin D-28K, S-100 proteins and calmodulin were localized in the Leydig cells, which are sites of testosterone synthesis. Only the appearance of parvalbumin-immunoreactivity is closely correlated to testosterone production during development of the testes. Calbindin D-28K-immunoreactivity persisted in foetal-type Leydig cells and in adult-type Leydig cells at all stages of development. S-100-immunoreactivity was low during all foetal stages, absent between birth and puberty, and increased thereafter. Calmodulin staining is most prominent in the cytoplasm of developing spermatocytes and of maturing spermatids. All four proteins co-exist in the seminiferous tubules. The distinct localization and developmental appearance of these proteins suggests different regulatory roles in Leydig cell function and spermatogenesis.

Animals↗
PubMedDate not supplied

In vitro interactions of calmodulin with the ovine proacrosin-acrosin system.

The authors studied the interaction of calmodulin (CaM) with proacrosin and acrosin from ram spermatozoa. CaM binding evaluated by the [125I]-CaM overlay procedure was shown to occur preferentially with both proacrosin and acrosin in the presence of EGTA; in the presence of Ca2+, the interaction was less intense. Further studies with native proenzyme preparations showed that proacrosin activation at pH 7.1 or 8.0 was significantly accelerated in the presence of CaM and EGTA (t1/2 = 23 min vs. 55 min for EGTA alone at pH 7.1), but not in the presence of Ca2+ (t1/2 = 73 min). The enzymatic activity of acrosin towards benzoyl arginine paranitroanilide, however, was not significantly affected by CaM whether Ca2+ was absent or present. Finally, the authors demonstrated that acrosin hydrolyzed CaM rapidly and extensively in the presence of EGTA. These results indicate that CaM interacts in vitro with proacrosin and acrosin, and that acrosin can attenuate CaM activity through proteolysis. Whether these interactions also occur in vivo and are involved in some aspects of spermatozoa function remains to be determined.

Acrosin↗