Biomedical subjects

K L Polakoski

Publications and source records attributed to K L Polakoski.

At least 19 recordsLinked to original sources

PubMed1997

The expression and activity of prostaglandin H synthase-2 is enhanced in trophoblast from women with preeclampsia.

Preeclampsia is associated with altered biosynthesis of vasoactive prostanoids in placental villi. The two isozymes of prostaglandin H synthase (PGHS) are essential for prostanoid synthesis. We tested the hypothesis that PGHS-2 expression is elevated in trophoblast from preeclamptic women, compared with trophoblast from healthy women. Using immunofluorescent staining, we demonstrated a higher PGHS-2 expression in villi from preeclampsia, compared with normal pregnancy. Cytotrophoblasts cultured from placentas of preeclamptic women expressed higher levels of PGHS-2 compared with cytotrophoblasts from normal placentas. This enhanced expression of PGHS-2 correlated with increased media levels of both thromboxane and prostaglandin E2, two products of PGHS activity. The increased prostanoid production by trophoblast from preeclamptic women was markedly reduced by NS-398, a specific inhibitor of PGHS-2. We conclude that both expression and activity of PGHS-2 are enhanced in trophoblasts from preeclamptic women compared with trophoblast from normal pregnancies. The increased production of prostanoids may contribute to the clinical syndrome of preeclampsia. Our data suggest that a selective inhibitor of PGHS-2 might provide a therapeutic alternative to prophylactic low-dose aspirin in modifying the prostanoid profile in preeclampsia.

Blotting, Northern↗

PubMed1995

Characterization of the heat shock protein P70 in rat spermatogenic cells.

A number of hsp70-like proteins are associated with developing male germ cells. One of these molecules, P70, is not sensitive to heat stress and is germ cell-specific, and its expression is developmentally regulated. We have characterized the association of the rat P70(rP70) with differentiating germ cells in the testis and with posttesticular sperm. An antibody originally raised against human sperm proacrosin (designated C3; Sigel et al., 1987: J Reprod Immunol 11:307-319) was found to immunostain rP70 by immunoblot analysis and was used in subsequent studies of the rP70 molecule. The C3 antibody reacted with P70 isoforms in rat, human, mouse, guinea pig, boar, and rooster testicular homogenates. In the developing rat testis, abundant rP70 protein levels were first detected on postnatal day 22, with upregulation to adult levels occurring after postnatal day 28. Purified populations of adult rat pachytene spermatocytes, round spermatids, and elongating spermatids, isolated by unit gravity velocity sedimentation, all expressed rP70. Posttesticular sperm exhibited a loss of the rP70 molecule; caput epididymal sperm were weakly immunoreactive for rP70, but no immunoreactivity was observed in either cauda epididymal sperm or epididymal fluid. In contrast to human ejaculated sperm, rat ejaculated sperm did not express rP70. The loss of P70 from rat posttesticular sperm may reflect species-specific differences in P70 functions, which are thought to include a role in the structural modifications that occur during germ cell differentiation.

Aged↗

PubMed1994

The effects of cocaine on neutral amino acid uptake by human placental basal membrane vesicles.

OBJECTIVE: Prior studies have demonstrated that cocaine binds to human placental microvillous membrane vesicles at a single high-affinity site and that both 10 and 500 nmol/L cocaine inhibit sodium-dependent alanine uptake. The purpose of this study was to characterize cocaine binding to human placental basal plasma membrane and to determine the effects of cocaine on basal vesicle uptake of alanine and leucine. STUDY DESIGN: Basal vesicles were isolated from the placentas of uncomplicated human pregnancies with no history of cocaine use. The binding of tritiated cocaine to basal vesicle membrane and the uptakes of tritiated cocaine, alanine, and leucine were determined with filtration assays. Alanine and leucine uptakes were measured in the presence and absence of sodium and 10 and 500 nmol/L cocaine. Cocaine binding was characterized with Scatchard analyses, and uptakes were compared by means of Student t tests. RESULTS: Tritiated cocaine bound to basal membrane at two separate high-affinity sites. Sodium-dependent alanine uptake was significantly inhibited only by 500 nmol/L cocaine. Sodium-independent amino acid uptake was unaffected by cocaine. CONCLUSION: Cocaine may interfere with fetal growth by impairing the activity of sodium-dependent amino acid transporters in both the microvillous and basal membrane. These membranes may be differentially sensitive to the effects of cocaine on such transporters.

Adult↗

PubMed1993

Cocaine inhibits alanine uptake by human placental microvillous membrane vesicles.

OBJECTIVE: The aim of this study was to determine the effects of cocaine on alanine uptake by human placental microvillous membrane vesicles and to characterize cocaine binding to the microvillous membrane. STUDY DESIGN: Microvillous vesicles were isolated from the placentas of 10 human pregnancies with no history of cocaine use. The binding of tritiated cocaine to microvillous vesicle membrane and uptake of tritiated cocaine and tritiated alanine were determined with the use of filtration assays. Scatchard analyses were used to characterize cocaine binding. Sodium-independent and sodium-dependent uptake of tritiated alanine was measured in the presence and absence of (-)cocaine and its stereoisomer (+)cocaine. Uptakes were compared with the use of Student t tests. RESULTS: Specific tritiated cocaine binding accounted for approximately 96% of total binding at a single-component high-affinity site in the microvillous membrane. The mediated sodium-dependent component of alanine uptake was significantly (p < 0.01) reduced in the presence of (-)cocaine but was unaffected by (+)cocaine. CONCLUSION: Cocaine may contribute to fetal growth restriction by interfering with the normal activity of placental amino acid transporters necessary to maintain the nutrient gradients associated with normal fetal growth.

Adult↗

PubMed1992

Demonstration of a boar testicular protein band that is immunoreactive to proacrosin and proacrosin binding protein antibodies.

A testicular protein band has been identified and shown to be immunoreactive to both of the proacrosin (53-55 kd) and the proacrosin binding protein (28 kd) antibodies. pH 4.5 extracts of boar testis were prepared and subjected to Western blot analysis using polyclonal antibodies of the proacrosin and the proacrosin binding protein. In addition to their respective antigens, a distinct high molecular weight protein band of approximately 200 kd was detected by both of the antibodies. Gelatin SDS-PAGE analysis of the extracts showed that this protein band was proteinase active. These results suggest that the proacrosin molecule is present as a much higher molecular weight form in the boar testis than the currently known 53-55 kd forms that have been isolated from spermatozoa.

Acrosin↗

PubMed1992

Proacrosin binding protein: immunocomparative studies in boar, bovine, hamster, human and ram.

The cross reactivities of acidic extracts from boar, bovine, hamster, human and ram spermatozoa to a polyclonal antibody of the boar proacrosin binding protein has been investigated. The pH 3.0 extracts of the washed spermatozoa from each species were subjected to Western blot analysis using a polyclonal antibody developed against the 28 kDa boar proacrosin binding protein. The boar sperm extracts had a major 28 kD and a minor 29 kDa proacrosin binding protein. A similar protein of 29 kDa was present in the bovine and ram samples, while the hamster had bands at 22 and 25 kDa. Extracts of human sperm yielded a diffuse but distinct area of cross reactivity of about 25-30 kDa. These results demonstrate that proacrosin binding proteins are present in sperm of different mammalian species and they have similar molecular weights.

Animals↗

PubMed1992

Distribution of tritiated cocaine in selected genital and nongenital organs following administration to male mice.

STUDY OBJECTIVE: To determine the distribution of cocaine administered to male mice in selected extragenital and genital organs and to investigate its possible binding to sperm. DESIGN: Twenty-seven sexually mature virus-free albino male mice were used in various experiments whereby following intravenous injection of tritiated cocaine hydrochloride, radioactivity was determined in several extragenital and genital organs, as well as sperm. RESULTS: Radioactivity was detected in all of the organs that were tested, and the highest concentrations per milligram of tissue were found in the kidney and epididymis. Removal of the sperm from the epididymis significantly reduced the radioactivity of the organ. The spermatozoa that were isolated on glass filters showed a linear correlation vs radioactivity (r = .93). CONCLUSIONS: Radioactivity is distributed to several organs, including the genital tract, and is found in association with sperm after in vivo administration of tritiated cocaine. These results may explain the mechanism underlying a male-mediated teratogenesis, which has been observed in animals that were exposed to cocaine, and they raise a possibility that the spermatozoa may carry cocaine into the oocyte during fertilization.

Animals↗

PubMed1991

Demonstration of specific binding of cocaine to human spermatozoa.

Exposure of males to cocaine has been linked to abnormal development of their offspring. To investigate the possible role of sperm, this study examined the interaction of cocaine with human spermatozoa. Washed sperm were incubated with tritiated cocaine (6.7 nmol/L) with or without unlabeled cocaine (670 mumol/L), and the samples were filtered and the remaining radioactivity quantitated. The specific binding was optimal at 20 minutes and 23 degrees C. Competition studies with tritiated cocaine (3.4 to 66.6 nmol/L) indicated the presence of approximately 3.6 x 10(3) binding sites per cell, with a high affinity receptor dissociation constant (Kd = 12.6 nmol/L). Cocaine concentrations as high as 670 mumol/L had no detectable effect on either the motility or viability of the cells. These results support the hypothesis that the sperm may act as a vector to transport cocaine into an ovum. This novel mechanism could be involved in the abnormal development of offspring of cocaine-exposed males.

Cocaine↗

PubMed1991

Identification of a proacrosin precursor in the cell-free translation of boar testicular poly(A)(+)-mRNA.

A cell-free translation system was used to determine the molecular mass of the protein component of precursor(s) to boar proacrosin. Poly(A)(+)-mRNA was extracted from freshly excised boar testis into phenol/chloroform, precipitated in chilled (-20 degrees C) ethanol, then translated in a cell-free, reticulocyte lysate system with Tran 35S-label. Analysis of the resulting products by SDS-PAGE followed by autoradiography demonstrated multiple bands of translated proteins. Both Western blotting and immunoprecipitation with a specific polyclonal antibody to boar proacrosin yielded a single major band with a relative molecular weight of approximately 64,000. These results suggest that proacrosin (Mr = 53,000-55,000), which contains both protein and carbohydrate moieties, results from the cellular processing of a proacrosin precursor molecule.

Acrosin↗

PubMed1990

Diisopropyl fluorophosphate labeling of sperm-associated proteinases.

Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.

Acrosin↗

PubMed1987

Biochemical and immunological comparisons between the human and boar proacrosin-acrosin proteinase systems.

Biochemical and immunochemical methods have been used to examine the proacrosin-acrosin system of human and boar spermatozoa. Marked biochemical similarities including the relative molecular weights of proacrosin (approx. 55,000), alpha-acrosin (45,000-49,000) and beta-acrosin (34,000-38,000) were observed for both species. In addition, the time course of proacrosin autoconversion between 0 to 60 min revealed that the purified proacrosin from both species autoconverted to alpha-acrosin and then to beta-acrosin at approximately the same time intervals. Despite these apparent biochemical similarities, distinct immunological differences between the human and boar proacrosin-acrosin systems were observed. The human proacrosin antibody immunoreacted with purified human proacrosin and alpha-acrosin but not with beta-acrosin. The antibodies to boar proacrosin cross-reacted with the purified boar proacrosin, alpha-acrosin and beta-acrosin. The antibodies to human proacrosin also cross-reacted with boar proacrosin and to a weak extent with boar alpha-acrosin but not with the boar beta-acrosin. While antibodies to boar proacrosin did not react with any of the components of the human proacrosin system. Additionally, in the non-purified sperm extracts the human proacrosin antibody preparation reacted with several proteins larger than proacrosin and one with a molecular weight of approximately 34,000. In the non-purified boar sperm extracts, the antibodies to boar proacrosin only cross-reacted with the known components of the proacrosin-acrosin system suggesting a high degree of specificity. Thus, immunochemical evidence is presented that indicates there are specific structural differences which occur in the proacrosin-acrosin system of mammalian sperm.

Acrosin↗

PubMed1987

Partial purification and characterization of human sperminogen.

A recently recognized non-proacrosin zymogen referred to as sperminogen has been purified from human spermatozoa, and several of its properties have been determined. The purification procedure included acid extraction of washed ejaculated sperm at pH 3.0, followed by gel filtration of the solubilized extract over a Sephadex G-75 superfine column. The sperminogen eluted from the column in a single band that was completely separated from the proacrosin band. This separation was confirmed by a gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) zymograph. This zymograph also demonstrated that the final sperminogen preparation contained four forms of zymogen, with molecular weights between 32,000 and 36,000. At neutral pH, the sperminogen was converted into spermin, its enzymatically active form, yielding a sigmoidal curve typical of zymogen autoactivation. The effects of several factors on the rate of this autoconversion indicate specific differences between sperminogen and proacrosin. Spermin hydrolyzed N-alpha-benzoyl-L-arginine ethyl ester (BzArgOEt), and was inhibited by lima bean trypsin inhibitor, pancreatic trypsin inhibitor, N-acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin), and tosyl-L-lysine chloromethyl ketone, indicating that the enzyme has a trypsin-like specificity and probably belongs to the class of trypsin-like enzymes. Since acrosin is generally believed to be the only trypsin-like enzyme in mammalian sperm, the demonstration of human sperminogen and spermin necessitates further inquiry into the functions and the relationships between sperm proteinase systems.

Acrosin↗

PubMed1986

The rapid purification and partial characterization of human sperm proacrosin using an automated fast protein liquid chromatography (FPLC) system.

A rapid and efficient procedure was developed for obtaining highly purified human proacrosin. Ejaculated spermatozoa were washed via centrifugation through 1 M sucrose containing 50 mM benzamidine and acid-extracted in the presence of benzamidine. The solubilized material was dialyzed then lyophilized. The sample was resuspended in 8 M guanidine hydrochloride in acetic acid (0.5 M) pH 2.5 and then subjected to gel permeation chromatography with an automated fast protein liquid chromatography system utilizing two Pharmacia Superose 12 columns set in tandem that were equilibrated in the same buffer. The proacrosin eluted as an individual peak that was well separated from another proteinase zymogen referred to as sperminogen. The proacrosin preparation was determined to be highly purified when observed on silver-stained SDS-polyacrylamide gels as well as on gelatin-SDS-polyacrylamide gels. The proacrosin appeared as a doublet (Mr = 55,000 and 53,000) on both of these systems. The autoconversion of proacrosin to acrosin at pH 8 resulted in a typical sigmoidal autoactivation curve. Following protein staining of SDS-polyacrylamide gels, it was shown that upon activation of purified proacrosin preparations the 55,000 and 53,000 molecular weight proteins were initially degraded to a 49,000 form and then to several lower molecular weight forms (Mr = 40,000-34,000). Similar findings with regard to proteolytic digestion were observed following gelatin-SDS-polyacrylamide zymography except that an increase with time in proteinase intensity between 58,000 and 53,000 was also observed. Cobalt and calcium were found to be potent inhibitors of the conversion of proacrosin into acrosin, while sodium resulted in much less inhibition of this process. Calcium was found to markedly enhance the proteolytic activity of human acrosin, while it had no observable influence on the acrosin hydrolysis of benzoylarginine ethyl ester. Thus, the described purification procedure resulted in a highly purified proacrosin preparation in sufficient yields to allow for its partial characterization.

Acrosin↗

PubMed1986

Quantification and partial characterization of the hamster sperm proacrosin-acrosin system.

The proacrosin-acrosin proteinase system was measured and partially characterized in unpurified extracts of washed hamster epididymal sperm. Autoactivation experiments demonstrated that proacrosin accounted for greater than 98% of the acrosin activity in the sperm extracts from individual animals. Several bands of proteinase activity were observed on gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic (gelatin-SDS-PAGE) zymography. The major proteinase activities in the nonactivated extracts corresponded to relative molecular masses (Mr) of 51,000 to 56,000, while less distinct digestion occurred with relative molecular masses of 37,000 to 49,000. It was demonstrated that after a serial dilution of the sperm extract, the proteinase activity in as few as 6,000 sperm could readily be detected by the gelatin-SDS-PAGE methods. Time-course activation studies showed that the zymogen was completely converted to active proteinase in 45-60 min at pH 8.0 and 25 degrees C. This autoconversion process was markedly inhibited by calcium, sodium, and heparin. However, each of these compounds stimulated the proteolytic activity of acrosin. These studies demonstrate that the proacrosin-acrosin system can be investigated in extracts of nonpurified hamster epididymal sperm.

Acrosin↗

PubMed1985

Evaluation of the human sperm proacrosin-acrosin system using gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophesis.

Proteolytic enzymes in extracts of human sperm have been identified and partially characterized using a technique which incorporates gelatin into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) system. Initially, semen characteristics from four donors were evaluated. Following this, washed sperm were acid extracted and proacrosin and acrosin activities determined spectrophotometrically. Proteinase activity in unactivated sperm extracts was then extracts was then demonstrated using the gelatin-SDS-PAGE system. Three major (Mr approximately equal to 47,000-54,000) and four faint (Mr approximately equal to 34,000-38,000) bands of digestion were observed. Upon activation of sperm extracts it was observed that maximum esterase activity occurred within 7 min of activation while maximum proteinase activity required approximately 15 min. When gels were washed and incubated in the presence of 50 mM benzamidine, no digestion bands were observed. This indicates that all of the digestion bands were due to trypsin-like proteinases. Finally, upon serial dilution of sperm extracts it was found that this SDS-PAGE system is sensitive enough to detect proteinase activity from as few as 30,000 sperm.

Acrosin↗

PubMed1983

Gossypol inhibition of acrosin and proacrosin, and oocyte penetration by human spermatozoa.

Gossypol, a known antispermatogenic agent, was found to effectively inhibit the highly purified boar sperm proacrosin-acrosin proteinase enzyme system by irreversibly preventing the autoproteolytic conversion of proacrosin to acrosin and reversibly inhibiting acrosin activity. The agent appears to prevent the self-catalyzed by not the acrosin-catalyzed activation of proacrosin. In additional experiments, brief exposure of human semen to concentrations of gossypol, which did not visibly alter spermatozoal motility or forward progression, was found to irreversibly inhibit the conversion of proacrosin to acrosin although the activity of the nonzymogen acrosin was not decreased, and also to prevent the human spermatozoa from penetrating denuded hamster oocytes. Gossypol inhibition of proacrosin conversion to acrosin closely paralleled the decline in oocyte penetration. Racemic (+/-) gossypol was equally as effective as the enantiomer (+) gossypol. The results suggest that the inhibition of proacrosin conversion to acrosin is a mechanism by which gossypol exerts its antifertility effect at nonspermicidal concentrations and that low levels of gossypol should be tested for their contraceptive action when placed vaginally.

Acrosin↗

PubMed1982

Acrosin inhibition. Comparisons of membrane-associated and -solubilized enzyme.

Acrosin is an extrinsic membrane proteinase from spermatozoa which functions in the fertilization process. Liposomes were utilized as a model system to determined possible effects of membrane association on acrosin's enzymatic activity. By comparison with solubilized enzyme, liposome-bound acrosin had a substantial reduction in the apparent affinity for "progressive" inhibitors such as leupeptin, lima bean trypsin inhibitor, soy bean trypsin inhibitor, and for a proteinase inhibitor from sperm extracts. In contrast, the liposome-bound and -solubilized enzymes were essentially identical with respect to the binding of benzamidine and p-aminobenzamidine which are competitive acrosin inhibitors. These results suggest membrane association can influence some but not all of acrosin's enzymatic properties.

Acrosin↗

PubMed1982

Proacrosin conversion inhibitor. Purification and initial characterization of a boar sperm protein which prevents the conversion of proacrosin into acrosin.

A proacrosin conversion inhibitor present in boar spermatozoa has been purified and initially characterized. Purification methods included sequential acid extractions of washed spermatozoa at pH 4.0, pH 3.5, and pH 2.5 followed by successive gel filtrations of the pH 2.5 sperm extract supernatant over Sephadex G-75 and G-50. The resulting 8.8-fold purified materials were judged to be homogeneous by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, had an estimated molecular weight of 12,800, and a constant specific activity of 65 units/mg. Treatment with the proteinases acrosin, trypsin, or chymotrypsin destroyed the highly purified proacrosin conversion inhibitor, indicating that it is a protein. Additional properties of the inhibitor included stability to long periods of storage at pH 3.0 and 4 degrees C, stability to boiling and lyophilization, and an absolute requirement for divalent cations to maintain activity. The highly purified proacrosin conversion inhibitor does not inhibit acrosin. Therefore, it apparently acts to prevent proacrosin conversion by selectively inhibiting the zymogen's self-catalyzed conversion mechanism.

Acrosin↗