Identification and expression of boar sperm proteins in the reproductive tract that interact with antibodies in serum from an infertile woman

Serum designated as IS obtained from a young healthy infertile woman induced a head-to-head agglutination of ejaculated boar sperm. The immunoglobulin G (IgG) prepared from IS localized to the acrosomal region of the sperm head obtained from the corpus and cauda epididymis as determined by an indirect immunofluorescent method. The IgG interacted with a boar sperm protein with an estimated molecular weight of 45-kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) immunoblotting technique. However, the IgG did not interact with proteins extracted from sperm obtained from the testis and caput epididymis or from non-gonadal tissues including liver, kidney, spleen, muscle and serum. The IgG interacted with additional proteins of about 75- and 38-kDa present in the corpus and cauda epididymal fluids but not those in the caput epididymal fluid. The staining intensity of the 75-kDa band was reduced and that of the 38-kDa was nullified with ejaculated seminal plasma proteins. The interacting proteins were adsorbed when chromatographed on Concanavalin A Sepharose column, suggesting that they are glycoproteins.     

Structural identity of immunoglobulin binding factor and prostatic secretory protein of human seminal plasma

The amino acid sequence of the N-terminus of the immunoglobulin binding factor of human seminal plasma was determined. The initial 30 amino acids showed complete identity with that of prostatic secretory protein, beta-microseminoprotein and beta-inhibin. In conclusion, these proteins are probably a single entity.     

Effect of auxotrophic mutation in Saccharomyces cerevisiae on the decay of intracellularly accumulated β-lactamase during vegetative growth,encoded on YEp vector

Summary Using aura3 ^– and a multi-marked yeastSaccharomyces cerevisiae strain, both of which carried YEp24, changes in -lactamase activity per ml of culture were monitored. This enzyme was subject to inactivation depending on growth phase; its degree was reduced by the presence of transformant-auxotrophy. The auxotroph (host: multi-marked) showed losses and quick recoveries of the activity during the approach to stationary phase, while the prototroph (host:ur3 ^–) less recovery after a dramatic loss. Physiological heterogeneity between auxotroph and prototroph, evaluated by comparing their growth parameters, might be responsible for such a marked contrast.     

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.     

A recB recC sbcB recJ host prevents recA-independent deletions in recombinant cosmid DNA propagated in Escherichia coli.

Segments of DNA are deleted from recombinant cosmid DNAs with high frequency during propagation in standard recA Escherichia coli hosts. An attempt has been made to derive an appropriate strain of E. coli, suitable for cosmid cloning, in which such deletions do not occur. We examined the effects of a series of host recombinational mutations on the deletion process, using six independent recombinant cosmids that carry inserts of mouse, Chinese hamster, or human DNA. Various E. coli host cells carrying the recombinant cosmids were cultured serially in liquid medium, and the recombinant cosmid DNAs were extracted from the host cells and analyzed by agarose gel electrophoresis and by gene transfer of the DNAs into cultured mammalian cells. Of the mutations examined, only a recB recC sbcB recJ (or recN) quadruple combination of host mutations prevented the deletion of DNA segments. The recombinant cosmid DNAs propagated in E. coli hosts that carried this combination of mutations were functionally as well as structurally intact. We propose that the recJ (and/or recN) gene is involved in some aspect of the events that lead to deletions of cosmid DNA in a recB recC sbcB genetic background.     

Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma

The sugar chains of transferrin samples, purified from sera of patients with hepatocellular carcinoma and of healthy individuals, were released quantitatively as radioactive oligosaccharides by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Comparative study of their structure by serial lectin column chromatography, by Bio-Gel P-4 column chromatography, and by sequential exoglycosidase digestion revealed that prominently altered glycosylation is commonly found in the hepatoma transferrins, although they all contain two complex-type asparagine-linked sugar chains in one molecule like in the case of normal transferrins. The alteration is quite various, including the increase of highly branched sugar chains, of those with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc beta 1----groups in their outer chain moieties and of those with a fucosylated trimannosyl core. Many but not all of the hepatoma transferrin samples contained a small amount of a bisected biantennary sugar chain, which was not detected in the normal transferrin samples.     

Effect of CS-514, a competitive inhibitor of hydroxymethylglutaryl coenzyme A reductase, on cholesterol gallstone formation in hamsters

Male golden hamsters fed a glucose diet as a model for cholesterol gallstone formation were used to investigate the effect of CS-514 on the lithogenicity of bile. Treatment with 0.05% (w/w) CS-514 in the diet for 1-4 weeks caused a decrease in plasma cholesterol and triacylglycerol levels. A marked increase in hepatic hydroxymethylglutaryl-CoA reductase activity in vitro and also an increased de novo cholesterol synthesis in the liver were induced by treatment with CS-514 for 1-4 weeks. The concentration of free cholesterol in liver microsomes and the cholesterol 7 alpha-hydroxylase activity were both decreased by treatment with CS-514 for 1 week, but were not affected by treatment for 4 weeks. The cholesterol output into bile and the lithogenic index of bile were double those of the control (glucose diet only) following treatment with CS-514 for 4 weeks, and the subsequent incidence of cholesterol gallstone formation was elevated. The content of free cholesterol and cholesterol ester in the liver was not affected by treatment with CS-514 for 4 weeks. These results suggest that long-term treatment with CS-514 causes a compensatory increase in the synthesis of hydroxymethylglutaryl-CoA reductase which leads to augmented hepatic de novo cholesterol synthesis and subsequent increased cholesterol output followed by an increase in the lithogenicity of bile. CS-514 apparently does not prevent cholesterol gallstone formation in those examples where the mechanism is thought to be due to augmented hepatic de novo cholesterol synthesis (type IV hyperlipidemia).     

Generation of Escherichia coli O9a Serotype, a Subtype of E. coli O9, by Transfer of the wb* Gene Cluster of Klebsiella O3 into E. coli via Recombination

Genetic characterization of the wb* gene in a series of Escherichia coli and Klebsiella strains possessing the mannose homopolymer as the O-specific polysaccharide was carried out. The partial nucleotide sequences and PCR-restriction fragment length polymorphism analysis suggested that E. coli serotype O9a, a subtype of E. coli O9, might have been generated by the insertion of the Klebsiella O3 wb* gene into a certain E. coli strain.     

Characterization of protein kinase C in Xenopus oocytes.

Protein kinase C (PKC) was partially purified from Xenopus laevis oocytes by ammonium sulfate fractionation followed by DEAE-cellulose and hydroxyapatite column chromatography. In the latter chromatography, two distinct PKC activities were identified. Both PKC fractions contained an 80 kDa protein which was recognized by three antisera raised against the conserved regions of mammalian PKC. However, specific antisera against alpha, beta I, beta II, and gamma-subspecies of rat PKC did not recognize the protein. Kinetic properties of the Xenopus PKCs were very similar to those of the rat alpha PKC, and only a subtle difference was found in the mode of activation by arachidonic acid. When oocytes were treated with the tumor promoter, phorbol 12-myristate 13-acetate, one of the Xenopus PKCs was found to disappear very rapidly, while the other remained unchanged up to 2 hr.     

Conformational equilibrium of an enzyme catalytic site in the allosteric transition.

The dynamic equilibrium of a catalytic site between active and inactive conformations, the missing link between the structure and function of allosteric enzymes, was identified using protein engineering and NMR techniques. Kinetic analyses of the wild-type and three mutants of Thermus L-lactate dehydrogenase established that the allosteric property of the enzyme is associated with a concerted transition between the high-affinity (R) and low-affinity (T) states. By introducing mutations, we prepared an enzyme in which the R and T states were balanced. The conformation of the enzyme-bound coenzyme, NAD+, which interacts directly with the substrate, was analyzed using NMR spectroscopy. NAD+ bound to the mutant enzyme was in a conformational mixture of the active and inactive forms, while NAD+ took on predominantly one of the two forms when it was bound to the other enzymes we had analyzed. We interpret this to mean that the catalytic site is in equilibrium between the two conformations. The ratio of the conformers of each enzyme agreed with the [T]/[R] ratio as determined by kinetic analyses. Therefore, it is the identified conformational equilibrium of the catalytic site that governs the allosteric regulation of the enzyme activity.