Comparative studies of elemental composition on ejaculated fowl, bull, rat, dog and boar spermatozoa by electron probe X-ray microanalysis

1. Comparative analyses of the concentrations of bulk and trace elements on the head, midpiece and tail regions of the ejaculated fowl, bull, rat, dog and boar spermatozoa were performed using X-ray microanalysis with scanning electron microscopy. 2. Although the pattern of distribution of elements on the surface of the three different subcellular regions was, in general, similar among all the species, there were substantial shifts in absolute concentrations. 3. Concentration of magnesium on the dog spermatozoa was significantly higher (about 2 times) than those of the other species. 4. Zinc, copper, iron and manganese concentrations were higher on fowl spermatozoa compared with those of the other species studied. 5. The Na-to-K ratios on the midpiece ranged from 1.46 (rat) to 2.26 (dog).     

Human p55(CDC)/Cdc20 associates with cyclin A and is phosphorylated by the cyclin A-Cdk2 complex

The initiation of anaphase and exit from mitosis depend on the activation of the anaphase-promoting complex/cyclosome (APC/C), a multicomponent, ubiquitin-protein ligase. The WD-repeat protein called p55(CDC)(Cdc20) directly binds to and activates APC/C. By using yeast two-hybrid screening, we found that cyclin A, a critical cell cycle regulator in the S and G2/M phases, specifically interacts with p55(CDC). Ectopically expressed p55(CDC) and cyclin A form a stable protein complex in mammalian cells. The p55(CDC)-cyclin A interaction occurs through the region containing the WD repeats of p55(CDC) and the region between the destruction box and the cyclin box of cyclin A. In addition to the physical interaction, p55(CDC) is phosphorylated by cyclin A-associated kinase. These findings suggest that the function of p55(CDC) is mediated or regulated by its complex formation with cyclin A.     

The possible role of protein-carboxyl methylation in the regulation of flagellar movement of fowl spermatozoa

Both intact and demembranated fowl spermatozoa were incubated at 30 degrees C and 40 degrees C with adenosine, 3-deazaadenosine and homocysteine thiolactone. This combination of products is known to block intracellular protein-carboxyl methylation reaction. The motility of intact spermatozoa incubated at 30 degrees C was vigorous but decreased markedly after the addition of 100 microM adenosine+100 microM 3-deazaadenosine+100 microM homocysteine thiolactone. During this incubation period, the intracellular ATP concentrations of spermatozoa were maintained at approximately 40 nmol ATP/10(9) cells, in spite of the inhibition of motility. The motility of demembranated spermatozoa at 30 degrees C was not inhibited by the same concentrations of blocker. At 40 degrees C, the motility of intact spermatozoa without any effectors was almost negligible. The addition of blocker did not appreciably affect the motility of spermatozoa, which remained almost negligible. In contrast, motility became vigorous even at 40 degrees C when intact spermatozoa were suspended in fluid to which had been added 1 mM CaCl(2) or 100 nM calyculin A, a specific inhibitor of protein phosphatase-type 1 and -type 2. Stimulation of motility by Ca(2+) or calyculin A was inhibited by the presence of a blocker. Contrary to that of intact spermatozoa, the motility of demembranated spermatozoa stimulated by protein phosphatase inhibitor at 40 degrees C was not inhibited by the presence of a blocker. These results suggest that protein-carboxyl methylation may be involved in the regulation of fowl sperm motility. Furthermore, it appears that the methylating enzyme may be present in the cytoplasmic matrix and/or the plasma membrane but not retained in the axoneme and/or accessory cytoskeletal components.     

In vitro effects of beta-carotene for the motility, ATP, and intracellular free Ca(2+) concentrations of fowl spermatozoa

The motility of intact fowl spermatozoa was vigorous at 25 degrees C, but decreased gradually following the addition of 0-100 microM beta-carotene in a dose-dependent manner. Even in the presence of stimulators of fowl sperm motility, such as Ca(2+) or calyculin A, the motility of intact spermatozoa at both 25 and 40 degrees C remained inhibited following the addition of beta-carotene. Under all of these circumstances, sperm ATP concentrations were not reduced by the addition of beta-carotene. Moreover, the motility of demembranated spermatozoa was not inhibited by the addition of the same concentrations of beta-carotene. No changes in intracellular free Ca(2+) concentrations, measured by means of a fluorescent Ca(2+) indicator, fura-2, were observed in intact beta-carotene -treated spermatozoa. These results suggest that beta-carotene is involved in the inhibition of the flagellar movement of fowl spermatozoa without change in energy production, and that the target of beta-carotene might be present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components.     

An enzymatic assay for myo-inositol in tissue samples

An enzymatic assay for myo-inositol (MI) was modified. The method is based on the oxidation of MI by NAD(+)-dependent MI dehydrogenase, coupled to reoxidation of NADH by iodonitrotetrazolium chloride and diaphorase. The resultant formazan is measured spectrophotometrically. In order to remove interference by glucose, preliminary phosphorylation of glucose by hexokinase was employed before the above reaction. The assay is quantitative for MI in amounts ranging from 1 to 20 nmol. This method gives a negligible blank, even in the measurement of rat serum. The reduced MI content in the sciatic nerve and lens of streptozotocin-induced diabetic rats recovered in a dose-dependent manner by treatment with a novel potent aldose reductase inhibitor, GP-1447 ?3-[(4,5, 7-trifluorobenzothiazol-2-yl)methyl]-5-methylphenylacetic acid?.     

Structural analysis of obscurin gene in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a cardiac disease characterized by left ventricular hypertrophy with diastolic dysfunction. Molecular genetic studies have revealed that HCM is caused by mutations in genes for sarcomere/Z-band components including titin/connectin and its associate proteins. However, disease-causing mutations can be found in about half of the patients, suggesting that other disease-causing genes remain to be identified. To explore a novel disease gene, we searched for obscurin gene (OBSCN) mutations in HCM patients, because obscurin interacts with titin/connectin. Two linked variants, Arg4344Gln and Ala4484Thr, were identified in a patient and functional analyses demonstrated that Arg4344Gln affected binding of obscurin to Z9-Z10 domains of titin/connectin, whereas Ala4484Thr did not. Myc-tagged obscurin showed that Arg4344Gln impaired obscurin localization to Z-band. These observations suggest that the obscurin abnormality may be involved in the pathogenesis of HCM.     

A sensitive and rapid in situ immunoassay to quantitatively determine the cellular antigens in intact cultured cells.

A sensitive and rapid in situ immunoassay to quantitatively determine the cellular antigens in intact cells was developed. Antigens located in plasma membrane, endosome, cytosol, lysosome or endoplasmic reticulum of cultured cells were fixed in situ and reacted with monoclonal antibodies after permeabilization of cells with saponin. The antigen-antibody complexes were quantified by colorimetric method of peroxidase-substrate reaction. Epidermal growth factor receptor, monomer of pyruvate kinase M2 and the endoplasmic reticulum-associated thyroid hormone-binding protein were easily detected from 0.5-1 x 10(4) cells by this method. Antibody as low as 10 ng/ml gave reproducible results. Using this method, the in vivo dynamic interconversion of monomer-tetramer of pyruvate kinase M2 was found to be regulated by glucose. The ligand-induced epidermal growth factor receptor through different subcellular compartments during endocytosis was easily quantified by this method. This method was also used to compare the different amounts of the endoplasmic reticulum-associated thyroid hormone-binding protein in various cultured cells. Thus, the in situ immunoassay is an easy and versatile method which can be used to study various cellular antigens and their involvement in cellular processes.     

Mechanism of DNA gyrase-mediated illegitimate recombination: characterization of Escherichia coli gyrA mutations that confer hyper-recombination phenotype.

To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we isolated temperature-sensitive gyrA mutants that confer spontaneous illegitimate recombination and spontaneous induction of lambda prophage at higher frequencies than that in the wild-type. After reconstruction of single mutations by targeted mutagenesis, we confirmed that two single mutations, gyrAL492P and gyrAL488P, and a double mutation, gyrAI203V+gyrAI205V, show the same properties as those described above. With respect to the phenotypes of hyper-recombination and higher induction of lambda prophage, these mutations were dominant over the wild-type. Analysis of recombination junctions of lambdabio transducing phages formed spontaneously in these mutants showed that the parental E. coli bio and lambda recombination sites have a homologous sequence of only 0. 7 base-pair on average, indicating that homology is not required for this illegitimate recombination. Analysis of nucleotide sequences of mutant gyrA genes revealed that the gyrAL492P and gyrAL488P mutations contain amino acid substitutions of Leu492-->Pro and Leu488-->Pro, respectively, which correspond to the alpha18 helix in the breakage-reunion domain of DNA gyrase A subunit. The gyrAI203V and gyrAI205V mutations contain Ile203-->Val and Ile205-->Val, respectively, which correspond to the alpha10' helix, also in the breakage-reunion domain of DNA gyrase A subunit. Biochemical analysis indicated that the GyrA63 protein that contains the L492P mutation has an apparently normal supercoiling activity, but it also produces a small amount of linear DNA in the absence of DNA gyrase inhibitor during the supercoiling reaction, suggesting that the mutant DNA gyrase may have a defect at the step of religation or a defect in the subunit interaction. These results suggest that the recombination is induced by defects of religation and/or dimer formation in the mutant DNA gyrases, implying that two alpha helices, alpha10' and alpha18, of DNA gyrase A subunit have crucial roles in subunit interaction and/or resealing of DNA.