Biomedical subjects

S S Koide

Publications and source records attributed to S S Koide.

At least 91 records · Page 5Linked to original sources

PubMed1990

Identification of hypoxanthine in bovine follicular fluid.

Bovine follicular fluid (bFF) blocks the occurrence of spontaneous germinal vesicle breakdown (GVBD) of isolated mouse oocytes. One of the active factors was purified by ultrafiltration through a PM-10 membrane filter, gel filtration through Sephadex G-25, and HPLC using a reversed-phase ODS-3 column and a Superose 12 column. This factor was identified as hypoxanthine by gas chromatography and mass spectrometry. The purified substance and hypoxanthine showed the same retention time on HPLC, and identical UV absorbance and mass spectra. There are other oocyte maturation inhibitors in follicular fluid that should be identified.

Animals↗

PubMed1990

Expression of a sperm protein gene during spermatogenesis in mammalian testis: an in situ hybridization study.

In previous work a specific membrane protein with an estimated Mr of 20.1 kDa was purified from rabbit sperm tails and designated as rSMP-B protein. Antibodies were raised against rSMP-B protein and used to isolate and identify the cDNA coding the rSMP-B protein from a rat testis lambda gt11 expression library. The nucleotide sequence of the cDNA was determined in a previous study. Single-stranded 35S-labeled RNA probes were prepared. With the techniques of in situ hybridization, rSMP-B mRNA was detected in spermatids of rat and rabbit testis. The present results support our previous observation that immunization of male rabbits with the rSMP-B protein results in the arrest of spermatogenesis at the spermatid stage. Overall, rSMP-B protein appears to be involved in spermiogenesis, and the synthesis of the mRNA encoding the protein occurs in germ cells during the postmeiotic haploid phase of spermatogenesis.

Animals↗

PubMed1990

Glycosaminoglycans in porcine follicular fluid promoting viability of oocytes in culture.

The viability of oocytes cultured in vitro was determined by the trypan blue exclusion test. Isolated porcine oocytes with or without cumulus cells cultured in modified Krebs-Ringer medium undergo cell death after 48 h. The addition of glycosaminoglycans (GAGs) prepared from porcine follicular fluid (pFF) to the medium delayed or prevented the onset of cell death in vitro. GAGs at concentrations of 0.25 mg/ml or greater prevented cell death in a dose-dependent manner. To identify the active factor, GAGs were purified from pFF by ethanol precipitation, chromatography on Dowex 1-x2, and high performance liquid chromatography (HPLC) on TSK gel DEAE-2 SW column. The fraction with a retention time nearly coincident with that of hyaluronic acid possessed high oocyte viability promoting activity. The present results suggest that the viability of oocytes in vitro is influenced by the presence of specific GAGs separated from follicular fluid.

Animals↗

PubMed1990

Characterization of cDNA encoding a human sperm membrane protein related to A4 amyloid protein.

A rat testis lambda gt11 cDNA library was screened with a monoclonal antibody raised against a human sperm membrane protein designated YWK-II. A clone was found with a cDNA insert composed of 1837 base pairs that contained an open reading frame coding for 191 amino acid residues. The deduced polypeptide contained a segment with high homology to the transmembrane-cytoplasmic domains of the A4 amyloid protein found in brain plaques of Alzheimer disease patients. A sequence of basic amino acid residues, Arg-Lys-Arg, was found instead of Lys-Lys-Lys at the probable membrane-cytoplasmic junction that may be a unique property of sperm membrane proteins.

Amino Acid Sequence↗

PubMed1990

Nucleic acid sequence of cDNA (RSD-1) encoding a rabbit sperm membrane protein and structural properties of the encoded protein.

The nucleotide sequence of a 2.0 kb cDNA encoding a rabbit sperm tail membrane protein was determined using the chain termination method. Analysis was performed using Sequenase and Klenow enzymes on the target sequence in an M13mp18/19 cloning system. The open reading frame consisted of 438 bp encoding 146 amino acids of the protein, with a calculated Mr of 15507. The hydrophobic amino acid content was 34.9%, and pI was in the vicinity of 5.8.

Amino Acid Sequence↗

PubMed1990

Inhibition of zygote formation and embryo development in mice immunized with monoclonal antisperm antibodies.

Passive immunization of female mice by the intraperitoneal administration of ascitic fluids containing monoclonal antisperm antibodies blocked the development of mouse zygotes to morulae and blastocysts when cultured in vitro. Radioiodinated YWK-I monoclonal antibodies were prepared and administered intraperitoneally to female mice. Significant radioactivity was found in washes of the oviductal lumens. The results suggest that antisperm antibodies block fertilization and may have a deleterious effect on early developing embryos.

Animals↗

PubMed1989

Transblot identification of biotin-containing proteins in rat liver.

Peroxidase-conjugated avidin was used to detect biotin-containing carboxylases in rat liver. By a transblot method, avidin-peroxidase interacted with liver proteins with estimated molecular masses of 120 and 74 kDa. The proteins were identified as pyruvate carboxylase (120 kDa, 6.4 pI) and methylcrotonyl-CoA carboxylase (74 kDa, 7.2 pI) by two-dimensional gel electrophoresis and transblot method. An additional band with estimated molecular mass of 220 kDa was detected in the cytosol fraction of rat liver, compatible with acetyl-CoA carboxylase. Rat liver proteins were prepared and treated with avidin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblot with avidin-peroxidase. A 190-kDa band was found with a parallel decrease in the 120-kDa band determined by Coomassie blue staining; however, these proteins did not stain by the transblot avidin-peroxidase method. When the transblot of parallel proteins was incubated with biotin and subsequently with avidin-peroxidase, two additional bands, namely 190 and 145 kDa, were detected while the 74-kDa band disappeared correlated with decreased staining of the 120-kDa band. The present procedure is a simple, rapid, and inexpensive method for detecting biotin-containing proteins in various tissues and organs and in determining the occurrence of nonspecific staining with the avidin-biotin complex method of immunoblot.

Animals↗

PubMed1989

Protein phosphorylation during 1-methyladenine-induced maturation of Asterias oocytes.

Maturation was induced in Asterias oocytes with 1-methyladenine (1-MA) at a final concentration of 2 microM. At 5, 10, and 30 min of treatment, oocytes were homogenized and the cytosolic fraction was prepared. The cytosol was incubated with [gamma-32P]ATP and [gamma-32P]GTP. The phosphorylated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the radioactivity in the gels was determined by autoradiography. The cytosol prepared from 1-MA-treated oocytes incubated with [gamma-32P]ATP showed a marked increase in the radiolabeling of proteins with estimated molecular weights of 70,000 and 62,000 Da. With [gamma-32P]GTP a 56,000-Da protein showed increased radiolabeling. The present finding suggests that an early biochemical event of 1-MA-induced oocyte maturation in Asterias is the stimulation of phosphorylation of specific proteins.

Adenine↗

PubMed1989

Carbohydrate moieties of small placental hCG: requirement of mannose structure for biological activity.

The 43 kDa human chorionic gonadotropin (hCG) (SP-hCG) was purified from human placenta and analyzed for sugar moieties. The low hexosamine content suggests that SP-hCG probably lacks O-linked sugar chains in the beta-subunit and incompletely formed N-linked sugar chains in the alpha- and beta-subunits. In the present study SP-hCG was hydrolyzed with various glycosidases. Treatment of hCG or SP-hCG with O-glycan peptide hydrolase increased the mobility of asialo-hCG beta in reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) while that of SP-hCG beta was unaffected, indicating that SP-hCG beta does not contain NeuNAc-Gal-GalNAc unit. Alpha-Mannosidase and endoglycosidase H hydrolyzed mannose and the high mannose-GlcNAc moieties, respectively, from alpha- and beta-subunits of SP-hCG, but not from the subunits of authentic hCG. Glycopeptidase F hydrolyzed completely the N-linked sugar chains from SP-hCG subunits, producing alpha- and beta-subunits with estimated Mr of 15,000 and 18,500, respectively. The biological activity of purified SP-hCG is about 50-80% of highly purified authentic hCG. In an in vitro system SP-hCG increased cAMP accumulation and testosterone production by rat Leydig cells to the same levels as that induced by hCG. However, the biological activity of SP-hCG was markedly reduced, following treatment with endoglycosidase H or alpha-mannosidase. To attain the level of testosterone production equivalent to that induced with untreated SP-hCG, 10-20 times higher dose of treated SP-hCG was required. On the other hand, cAMP accumulation induced with treated SP-hCG even at a very high concentration was substantially lower than that attained with untreated SP-hCG. In conclusion, the mannose moieties are essential structural components of the hormone in stimulating cAMP accumulation and steroidogenesis by rat Leydig cells.

Animals↗

PubMed1988

Phosphorylation activity associated with plasma membrane-cortical granule complex of Xenopus laevis oocytes.

Plasma membrane-cortical granule complexes from Xenopus laevis oocytes were isolated by a differential centrifugation procedure. Membrane preparations incorporated 32P from [gamma-32P]ATP predominantly into three protein bands of Mr 128,000, 54,000 and 29,000, as determined by sodium dodecyl-polyacrylamide gel electrophoresis. Progesterone and insulin, added in vitro to membranes, did not produce any change in this phosphorylation pattern, contrary to some previously published results. The availability of enriched membrane complexes should permit further biochemical dissection of signal transduction pathways that are activated during oocyte maturation and fertilization.

Animals↗

PubMed1988

Isolation and partial characterization of an ion channel protein from human sperm membranes.

Human sperm cells were fractionated and plasma membrane proteins were separated by molecular gel sieving chromatography (Sephacryl S-200 followed by HPLC). A pore-forming protein was extracted from sperm cell membranes. The partially purified protein migrated with Mr 100,000-110,000, as determined by molecular sieving gel chromatography, and with a Mr 90,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The channel activity was also extracted with Triton X-114, suggesting a hydrophobic nature for this protein. This protein was incorporated into planar lipid bilayers, resulting in the formation of voltage-dependent ion channels. Single channel fluctuations of 130 pS/unit in 0.1 M NaCl were resolved; however, channels preferentially aggregated in triplets having an open state life-time that persisted for several seconds. The channels studied here were more selective for monovalent cations than anions, but also showed some permeability to anions and larger electrolytes, suggesting a large functional pore diameter. The role of this sperm channel in normal sperm physiology and/or fertilization is presently unclear.

Cell Membrane Permeability↗

PubMed1988

Identification of cell types in Sertoli cell-enriched cultures by immunocytochemistry and DNA-specific fluorochrome Hoechst 33342.

The cell types in Sertoli cell-enriched cultures can be identified by using the DNA-specific fluorochrome Hoechst 33342 staining. This simple, rapid and reproducible procedure can be used with fixed and living cells. The peritubular myoid cells can be distinguished from the Sertoli cells in Sertoli cell-enriched cultures by the characteristic staining pattern obtained using Hoechst 33342 dye. Those cells identified as peritubular myoid cells by the characteristic DNA staining also interacted with the anti-fibronectin antibody determined by an immunocytochemical method while the Sertoli cells did not. The described staining method is valuable in assessing the presence of peritubular myoid cells in Sertoli cell-enriched cultures.

Animals↗

PubMed1988

Interaction of gossypol with sperm macromolecules and enzymes.

The interaction of gossypol with sperm macromolecules is an important process for the gossypol action. It is of our interests to study the nature of this interaction in order to understand the mechanism of action of gossypol. Our experimental results support the thesis that gossypol inhibits sperm motility by blocking ATP production and utilization. The drug acts on the mitochondria, suppressing oxygen consumption, inhibiting pyruvate dehydrogenase and ATPase activities and probably on the motility apparatus by blocking dynein ATPase activity and preventing protein phosphorylation. The nature of the chemical bond in the interaction of gossypol and macromolecules is through the Schiff base formation and by the hydrophobic and nucleophilic attraction.

Adenosine Triphosphatases↗

PubMed1988

Protein phosphorylation during 5-hydroxytryptamine-induced maturation of Spisula oocytes.

Maturation was induced in Spisula oocytes with 5-hydroxytryptamine (5-HT) creatinine sulfate at a final concentration of 5 microM. After 10 and 30 min of treatment, oocytes were homogenized and the cytosolic and particulate fractions were prepared. The fractions were incubated with [gamma-32P]GTP and [gamma-32P]ATP. The phosphorylated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactivity in the gels was determined by autoradiography. With [gamma-32P]GTP a marked increase in the radiolabeling of proteins with an estimated Mr of 47,000 and 20,000 in the cytosolic and particulate fractions, respectively, was demonstrated with the 5-HT-treated oocytes, whereas no stimulation was demonstrable with the use of [gamma-32P]ATP. A significant increase in GTP-mediated protein phosphorylation occurred within 10 min after 5-HT treatment before the occurrence of germinal vesicle breakdown, suggesting that this post-translation modification of proteins is an early action of the neurotransmitter in the induction of meiotic reinitiation in oocytes.

Adenosine Triphosphate↗

PubMed1988

Purification and characterization of an incompletely glycosylated form of human chorionic gonadotropin from human placenta.

A small form of hCG (SP-hCG) was purified from an acetone powder preparation of human first trimester placenta by repeated gel filtration and ion exchange chromatography on a Q-Sepharose or FPLC Mono Q column. The estimated mol wt (Mr) of the small hCG by gel filtration is 43K compared to 58K for authentic hCG. The pI of SP-hCG is 10.0, suggesting deficiency of sialic acids. SP-hCG dissociates into subunits when treated with 6 M guanidine-HCl or analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two beta-subunits of SP-hCG were found with estimated Mr of 23K and 20K. Both are distinctly smaller than authentic hCG beta. A single alpha-subunit was found, with an estimated Mr of 21 K. The immunoactivity (8,900-10,000 IU/mg) of highly purified SP-hCG was comparable to that of reference hCG (CR119) determined by a RIA method using anti-hCG antibodies. The hCG/LH receptor-binding activity of SP-hCG is equivalent to that of reference hCG (CR119). Its biological activity is lower than that of reference hCG (approximately 30% or more) assayed by the in vitro stimulation of rat Leydig cells to produce testosterone and cAMP. A high dose is required to attain the same level of stimulation as reference hCG. The amino acid composition of SP-hCG is similar to that of reference hCG, whereas its hexsamine content is significantly lower. Its glucosamine content is about half that in reference hCG, while it completely lacks galactosamine. These findings suggest that SP-hCG is deficient in O-linked oligosaccharide chain in the beta-subunit, and that the N-linked oligosaccharide chains of both subunits are shortened. SP-hCG is one of the principal forms of the hormone present in first trimester placenta and may be a key intermediate in the posttranslational biosynthesis of hCG. Although it lacks O-linked sugar chains and shortened N-linked sugar chains, it possesses substantial biological activity. To have full biological activity, the hCG molecule must contain the complete complement of sugar chains.

Amino Acids↗

PubMed1988

Characteristics of the inhibition of poly(ADP-ribose) glycohydrolase by homopolypurines.

Poly(ADP-ribose) glycohydrolase partially purified from rat testis was markedly inhibited by the homopolypurines polyG, polyI and polyA. The inhibition was competitive with respect to poly(ADP-ribose) and the Ki for polyG and polyA was 2.8 uM and 5.5 uM, respectively. This inhibitory effect of the homopolypurines was practically eliminated when 250 mM KCl was present in the reaction mixture. Moreover, the inhibition exerted by polyI or polyA was markedly diminished after hybridization with polyC or polyT, respectively.

Animals↗

PubMed1987

Poly (ADP-ribose) synthetase is phosphorylated by protein kinase C in vitro.

Poly (ADP-ribose) synthetase from bovine thymus was phosphorylated effectively by protein kinase C in vitro. The phosphorylation was dependent on the activators of this kinase, Ca2+ and phospholipid. The apparent Km for the synthetase was about 8 microM, which was lower than that for histone H1. Though the synthetase was a weak substrate for Ca2+/calmodulin-dependent protein kinase II, other protein kinases, cyclic AMP-dependent and cofactor-independent protein kinases did not phosphorylate the synthetase. Phosphorylation of the synthetase by protein kinase C resulted in appreciable inhibition of the synthetase activity.

Animals↗

PubMed1987

A factor potentiating serotonin in the induction of germinal vesicle breakdown in surf clam oocytes.

Simultaneous addition of an aliquot of body fluid obtained from the surf clam, Spisula solidissima, enhanced oocyte germinal vesicle breakdown induced with serotonin but not with KCl. When the body fluid and serotonin were added sequentially to the oocytes, potentiation did not occur. Body fluids of both males and females were effective at a 200-fold dilution. The factor is stable when treated with heat, acid, base, trypsin and pronase. It is hydrophobic and not dialyzable through tubing with a molecular weight cutoff of 1000 daltons. The factor is probably not a protein.

Animals↗