Inwardly rectifying K(+) channels in spermatogenic cells: functional expression and implication in sperm capacitation

To fertilize, mammalian sperm must complete a maturational process called capacitation. It is thought that the membrane potential of sperm hyperpolarizes during capacitation, possibly due to the opening of K(+) channels, but electrophysiological evidence is lacking. In this report, using patch-clamp recordings obtained from isolated mouse spermatogenic cells we document the presence of a novel K(+)-selective inwardly rectifying current. Macroscopic current activated at membrane potentials below the equilibrium potential for K(+) and its magnitude was dependent on the external K(+) concentration. The channels selected K(+) over other monovalent cations. Current was virtually absent when external K(+) was replaced with Na(+) or N-methyl-D-glucamine. Addition of Cs(+) or Ba(2+) (IC(50) of approximately 15 microM) to the external solution effectively blocked K(+) current. Dialyzing the cells with a Mg(2+)-free solution did not affect channel activity. Cytosolic acidification reversibly inhibited the current. We verified that the resting membrane potential of mouse sperm changed from -52 +/- 6 to -66 +/- 9 mV during capacitation in vitro. Notably, application of 0.3-1 mM Ba(2+) during capacitation prevented this hyperpolarization and decreased the subsequent exocytotic response to zona pellucida. A mechanism is proposed whereby opening of inwardly rectifying K(+) channels may produce hyperpolarization under physiological conditions and contribute to the cellular changes that give rise to the capacitated state in mature sperm.     

Two isoforms of a nucleotide-sugar pyrophosphatase/phosphodiesterase from barley leaves (Hordeum vulgare L.) are distinct oligomers of HvGLP1, a germin-like protein

Two isoforms of ADPglucose pyrophosphatase/phosphodiesterase (AGPPase) have been characterized using barley leaves (Hordeum vulgare L.). Whilst one of the isoforms, designated as soluble AGPPase1 (SAGPPase1), is soluble in low ionic strength buffers, the other, SAGPPase2, is extractable using cell wall hydrolytic enzymes or high salt concentration solutions, thus indicating that it is adventitiously bound to the cell wall. Both AGPPase isoforms are highly resistant to SDS, this characteristic being utilized to purify them to homogeneity after zymographic detection of AGPPase activity in SDS-containing gels. N-terminal and internal amino acid sequencing analyses revealed that both SAGPPase1 and SAGPPase2 are distinct oligomers of the previously designated HvGLP1, which is a member of the ubiquitously distributed group of proteins of unknown function designated as germin-like proteins (GLPs).     

ESR studies on reaction of saccharide with the free radicals generated from the xanthine oxidase/hypoxanthine system containing iron

The free radicals generated from the iron containing system of xanthine oxidase and hypoxanthine (Fe-XO/HX) were directly detected by using spin trapping. It was found that not only superoxide anion (O(2)*-) and hydroxyl radical (OH*), but also alkyl or alkoxyl radicals (R*) were formed when saccharides such as glucose, fructose and sucrose were added into the Fe-XO/HX system. The generated amount of R* was dependent on the kind and concentration of saccharides added into the Fe-XO/HX system and no R* were detected in the absence of saccharides, indicating that there is an interaction between the saccharide molecules and the free radicals generated from the Fe-XO/HX system and saccharide molecules are essential for generating R* in the Fe-XO/HX system. It is expected that the toxicity of R* would be greater than of hydrophilic O(2)*- and OH* because they are liposoluble and their lives are longer and the active sites of biomolecules are closely related with lipophilic phase, thus they can damage cells more seriously than O(2)*- and OH*. The R* generated from the saccharide containing Fe-XO/HX can be effectively scavenged by selenium containing abzyme (Se-abzyme), indicating Se-abzyme is a promising antioxidant.     

Interplay between hydrophobic cluster and loop propensity in beta-hairpin formation

Autonomously folding beta-hairpins have recently emerged as powerful tools for elucidating the origins of antiparallel beta-sheet folding preferences. Analysis of such model systems has suggested four potential sources of beta-sheet stability: (1) the conformational propensity of the loop segment that connects adjacent strands; (2) favorable contacts between side-chains on adjacent strands; (3) interstrand hydrogen bonds; and (4) the intrinsic beta-sheet propensities of the strand residues. We describe the design and analysis of a series of isomeric 20 residue peptides in which factors (1)-(4) are identical. Differences in beta-hairpin formation within this series demonstrate that these four factors, individually, are not sufficient to explain beta-sheet stability. In agreement with the prediction of a simple statistical mechanical model for beta-hairpin formation, our results show that the separation between the loop segment and an interstrand cluster of hydrophobic side-chains strongly influences beta-hairpin size and stability, with a smaller separation leading to greater stability.     

Analysis of the fatty acid components in a perchloric acid-soluble protein

We had previously found that a perchloric acid-soluble protein (PSP1) occurs in rat liver, and that this novel protein inhibits protein synthesis in a rabbit reticulocyte lysate system (T. Oka, H. Tsuji, C. Noda, K. Sakai, Y.-H. Hong, I. Suzuki, S. Mu?oz, Y. Natori, J. Biol. Chem. 270 (1995) 30060-30067). In the present study, we analyzed lipid components bound to PSP1. Native PSP1 was purified from rat liver using Sephadex G-75, DE-52 cellulose and IgGPSP-affinity chromatography, and the lipid components were extracted. The components obtained from the purified PSP1 were shown to be free fatty acids by thin-layer chromatography. By GC-MS, six major fatty acids were identified as 14:0, 16:0, 18:0, 18:1, 18:2 and 20:4. 1 mol of PSP1 contained 1.26 mol of total fatty acid components. The fatty acid-binding assay of PSP1 showed that the Bmax was 1.25 mol fatty acid/mol PSP1 and the Kd value for palmitic acid was 6.03 microM. The concentration of PSP1 mRNA in rat liver increased 2.3-fold by the administration of peroxisome proliferator, bezafibrate. These findings show that PSP1 is a fatty acid-binding protein-like protein, which is involved in the intracellular metabolism of fatty acid and is quite different from the known fatty acid-binding proteins.     

Scedosporium apiospermum external otitis.

We report a case of Scedosporium apiospermum external otitis. The patient was topically treated with miconazole cream and achieved a clinical and mycological cure. The etiology, diagnosis and treatment of external fungal otitis are discussed.     

First molecular characterization of a uridine diphosphate galacturonate 4-epimerase: an enzyme required for capsular biosynthesis in Streptococcus pneumoniae type 1

Uridine diphosphate galacturonate 4-epimerases (UDPGLEs) are enzymes that convert UDP-glucuronate into UDP-galacturonate. Although the presence of UDPGLEs has been reported in prokaryoic and eukaryotic organisms, the genes coding for these enzymes are completely unknown. The galacturonic acid-containing capsular polysaccharide of Streptococcus pneumoniae type 1 is synthesized through the action of a specific UDPGLE. We have constructed a defined deletion mutant in the cap1J gene (one of the 15 cap1 genes responsible for the synthesis of the type 1 capsule) that exhibited an unencapsulated phenotype. This mutant was unable to synthesize UDPGLE, suggesting that Cap1J was the type 1-specific UDPGLE of S. pneumoniae. Escherichia coli cells harbouring the recombinant plasmid pRMM38 (cap1J) overproduced a 40 kDa protein, characterized as Cap1J on the basis of the N-terminal amino acid sequence analysis, and expressed high levels of enzymatically active Cap1J epimerase. Cap1J was partially purified, although purification to electrophoretic homogeneity inactivated the enzyme irreversibly. The enzyme has the following characteristics: K(m) for UDP-glucuronate, 0.24 mM; pH optimum, 7.5; equilibrium constant (in the direction of UDP-galacturonate formation), 1.3; and an approximate M(r) of 80,000 for the active form. The Cap1J protein exhibited a fluorescence emission spectrum similar to that of NADH. Upon inactivation with p-hydroxymercuribenzoate, the addition of NAD+ and 2-mercaptoethanol were sufficient to reactivate the enzyme. Among several compounds tested, UDP-galactose and UDP-xylose exhibited the highest inhibition of the UDPGLE activity. Inactivation of UDPGLE activity was also observed in the presence of UMP and several reducing sugars. To our knowledge, this is the first example of a thoroughly molecular characterization of a UDPGLE.     

The identification of Wos2, a p23 homologue that interacts with Wee1 and Cdc2 in the mitotic control of fission yeasts

The Wee1 kinase inhibits entry into mitosis by phosphorylation of the Cdc2 kinase. Searching for multicopy suppressors that abolish this inhibition in the fission yeast, we have identified a novel gene, here named wos2, encoding a protein with significant homology to human p23, an Hsp90-associated cochaperone. The deletion mutant has a modest phenotype, being heat-shock sensitive. Using antibodies raised against bacterially produced protein, we determined that Wos2 is very abundant, ubiquitously distributed in the yeast cell, and its expression dropped drastically as cells entered into early stationary phase, indicating that its function is associated with cell proliferation. In proliferating cells, the amount of Wos2 protein was not subjected to cell cycle regulation. However, in vitro assays demonstrated that this Hsp90 cochaperone is potentially regulated by phosphorylation. In addition to suppressing Wee1 activity, overproduction of Wos2 displayed synthetic lethality with Cdc2 mutant proteins, indicating that this Hsp90 cochaperone functionally interacts with Cdc2. The level of Cdc2 protein and its associated H1 kinase activity under synthetic lethal conditions suggested a regulatory role for this Wos2-Cdc2 interaction. Hsp90 complexes are required for CDK regulation; the synergy found between the excess of Wos2 and a deficiency in Hsp90 activity suggests that Wos2 could specifically interfere with the Hsp90-dependent regulation of Cdc2. In vitro analysis indicated that the above genetic interactions could take place by physical association of Wos2 with the single CDK complex of the fission yeast. Expression of the budding yeast p23 protein (encoded by the SBA1 gene) in the fission yeast indicated that Wos2 and Sba1 are functionally exchangeable and therefore that properties described here for Wos2 could be of wide significance in understanding the biological function of cochaperone p23 in eukaryotic cells.     

Effect of weaning time and associated management practices on postweaning chronic diarrhea in captive rhesus monkeys (Macaca mulatta)

OBJECTIVE: Our purpose was to assess the extent to which early weaning and other weaning-management factors affect development of postweaning chronic diarrhea in captive rhesus monkeys at the California Regional Primate Research Center between 1992 and 1995. METHODS: Data for weaning, management, and onset of diarrhea were obtained from daily records. The Cox proportional hazard model was used to assess whether the risk of chronic diarrhea was related to early weaning. RESULTS: Monkeys that were lighter at weaning had a threefold increase in risk of postweaning chronic diarrhea (P = 0.07), compared with that in heavier monkeys. An episode of preweaning diarrhea increased the risk of postweaning chronic diarrhea twofold (P = 0.08). Relocation of monkeys to outdoor facilities in the fall was associated with a fivefold decrease in risk (P < 0.001), compared with that of other seasons, and weaning in 1993 was associated with a twofold decrease in risk, compared with that of other years (P = 0.04). CONCLUSIONS: Multiple factors need to be considered for prevention of postweaning chronic diarrhea, including weaning weight, preweaning diarrhea, season weaned, and weaning conditions that change from year to year.     

A fluorescence study of the molecular interactions of harmane with the nucleobases, their nucleosides and mononucleotides

Fluorescence binding studies of harmane to the elemental components of the nucleic acids were undertaken to investigate the origin of the interaction between the drug and DNA. Most of the tested substrates have been found to induce hypochromism in the absorption spectrum of harmane and to quench its fluorescence. The quenching process induced by the nucleobases and their nucleosides is mainly due to the formation of ground state 1:1 complexes. However, in the case of the mononucleotides a dynamic quenching component is also observed. This quenching component is likely due to the excited state interaction of harmane with the phosphate group of the nucleotides. UV-vis spectral changes and quenching measurements have been used to quantify the ground state association constants of the complexes and the quenching rate constants.