Inference of a genetic network by a combined approach of cluster analysis and graphical Gaussian modeling

MOTIVATION: Recent advances in DNA microarray technologies have made it possible to measure the expression levels of thousands of genes simultaneously under different conditions. The data obtained by microarray analyses are called expression profile data. One type of important information underlying the expression profile data is the 'genetic network,' that is, the regulatory network among genes. Graphical Gaussian Modeling (GGM) is a widely utilized method to infer or test relationships among a plural of variables. RESULTS: In this study, we developed a method combining the cluster analysis with GGM for the inference of the genetic network from the expression profile data. The expression profile data of 2467 Saccharomyces cerevisiae genes measured under 79 different conditions (Eisen et al., PROC: Natl Acad. Sci. USA, 95, 14683-14868, 1998) were used for this study. At first, the 2467 genes were classified into 34 clusters by a cluster analysis, as a preprocessing for GGM. Then, the expression levels of the genes in each cluster were averaged for each condition. The averaged expression profile data of 34 clusters were subjected to GGM, and a partial correlation coefficient matrix was obtained as a model of the genetic network of S. cerevisiae. The accuracy of the inferred network was examined by the agreement of our results with the cumulative results of experimental studies.     

The prototype of sex chromosomes found in Korean populations of Rana rugosa

The seventh largest chromosome in Japanese populations of the frog Rana rugosa morphologically evolved as a sex chromosome. The sex chromosome is XX/XY type in one geographic form and ZZ/ZW type in another. In contrast, the seventh chromosomes are still homomorphic between the sexes in the other two geographic forms: they are more subtelocentric in the Kanto form and subtelocentric in the western Japanese form. To identify a prototype of the sex chromosomes, we extended our investigation in this study to the Korean form, which is supposed to be close to the phylogenetic origin of this species. The karyotype, a sex-linked gene sequence, and mechanisms of sex determination and gonadal differentiation were all examined. In addition, phylogenetic analyses were performed based on mitochondrial gene sequences and the results of crossings between the Korean and Japanese forms. As a consequence, the more subtelocentric seventh chromosome, shared by the Korean and Japanese Kanto forms, was concluded to be the prototype of the sex chromosomes. Starting at the prototype, a whole process of morphological sex chromosome evolution was reconstructed.     

A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease

A novel gram-positive, strictly aerobic, motile, sporulating, and facultatively alkaliphilic bacterium designated KSM-KP43 was isolated from a sample of soil. The results of 16S rRNA sequence analysis placed this bacterium in a cluster with Bacillus halmapalus. However, the level of the DNA-DNA hybridization of KSM-KP43 with B. halmapalus was less than 25%. Moreover, the G + C contents of the genomic DNA were 41.6 mol% for KSM-KP43 and 38.6 mol% for B. halmapalus. Because there were also differences in physiological properties and cellular fatty acid composition between the two organisms, we propose KSM-KP43 as a novel species of alkaliphilic Bacillus. This novel strain produces a new class of protease, an oxidatively stable serine protease that is suitable for use in bleach-based detergents. The enzyme contained 640 amino acid residues, including a possible approximately 200-amino-acid prepropeptide in the N-terminal and a unique stretch of approximately 160 amino acids in the C-terminal regions (434-amino-acid mature enzyme with a calculated molecular mass of 45,301 Da). The C-terminal half after the putative catalytic Ser255 and the contiguous C-terminal extension shared local similarity to internal segments of a membrane-associated serine protease of a marine microbial assemblage and the serine protease/ABC transporter precursors of the slime mold Dictyostelium discoideum, and to the C-terminal half of a cold-active alkaline serine protease of a psychrotrophic Shewanella strain.     

Metabolism of carcinogenic urethane to nitric oxide is involved in oxidative DNA damage

Carcinogenic urethane (ethyl carbamate) forms DNA adduct via epoxide, whereas carcinogenic methyl carbamate can not. To clarify a mechanism independent of DNA adduct formation, we examined DNA damage induced by N-hydroxyurethane, a urethane metabolite, using 32P-5'-end-labeled DNA fragments. N-hydroxyurethane induced Cu(II)-mediated DNA damage especially at thymine and cytosine residues. DNA damage was inhibited by both catalase and bathocuproine, suggesting a role for H(2)O(2) and Cu(I) in DNA damage. Free (*) OH scavengers did not inhibit the DNA damage, although methional did inhibit it. These results suggest that reactive species, such as the Cu(I)-hydroperoxo complex, cause DNA damage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was increased by N-hydroxyurethane in the presence of Cu(II). When treated with esterase, N-hydroxyurethane induced 8-oxodG formation to a similar extent as that induced by hydroxylamine. Enhancement of DNA cleavages by endonuclease IV suggests that hydroxylamine induced depurination. Furthermore, hydroxylamine induced a significant increase in 8-oxodG formation in HL-60 cells but not in its H(2)O(2)-resistant clone HP 100 cells. o-Phenanthroline significantly inhibited the 8-oxodG formation in HL-60 cells, confirming the involvement of metal ions in the 8-oxodG formation by hydroxylamine. Electron spin resonance spectroscopy, utilizing Fe[N-(dithiocarboxy)sarcosine](3), demonstrated that nitric oxide (NO) was generated from hydroxylamine and esterase-treated N-hydroxyurethane. It is concluded that urethane may induce carcinogenesis through oxidation and, to a lesser extent, depurination of DNA by its metabolites.     

Isolation of the interacting molecules with GEX-3 by a novel functional screening

To screen for important molecules that interact with a gene of interest in Caenorhabditis elegans (C. elegans), we established a novel functional screening system using the yeast two-hybrid system with the RNA interference technique. Our screening system makes it possible to identify the molecular machinery involved in the function of a gene of interest starting with the cDNA of this gene. As a model case, we examined the molecular machinery involved in the function of GEX-3, an essential factor of tissue morphogenesis. We identified many interacting molecules by yeast two-hybrid screening and could detect some functional interactions using this novel functional screening system.     

Approximation equation for oxygen uptake kinetics in decrement-load exercise starting from low exercise intensity

The purpose of the present study was to determine the degree of fitting an approximation equation for oxygen uptake (Vo(2)) in decrement-load exercise (DLE). Work rate was started from 120 watts and was decreased by a rate of 15 watts per min. The initial work rate of DLE corresponded to 72+/-10% of the work rate at anaerobic threshold determined in incremental-load exercise (ILE). Vo(2) in DLE increased rapidly, reached a peak, and decreased linearly until the end of the exercise. Vo(2) in DLE was higher than that in ILE at the same work rate except in the early periods in ILE and DLE. This difference ranged from 300 to 400 ml/min. This difference is a result of repayment of oxygen debt in DLE and from the oxygen deficit induced by the delay of response of Vo(2) in ILE. As work rate in DLE can be obtained by the difference between work rates in constant-load exercise (CLE) and ILE, we postulated that the approximation equation for Vo(2) kinetics in DLE could be expressed by a combination of approximation equations in CLE and in ILE. When time delay was taken into consideration in this equation, the fitting of data obtained by using the equation was better than that of data obtained by using the equation without a parameter of time delay. The degree of fitting ranged from 94 to 98% (r(2)). Thus, it seems that Vo(2) including oxygen debt in DLE can be approximated by the equation used in this study.     

Tyrosine phosphorylation is involved in Ca(2+)entry in human gingival fibroblasts

Bradykinin (1 microM) and histamine (100 microM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca(2+) release from intracellular stores and Ca(2+) entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 microM) and tyrphostin 47 (20 microM), the sustained level of [Ca(2+)](i) induced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca(2+), bradykinin and histamine induced only the transient increase in [Ca(2+)](i), but a subsequent addition of Ca(2+) to the medium resulted in a sustained increase in [Ca(2+)](i) caused by Ca(2+)entry. Thapsigargin, an inhibitor of Ca(2+)-ATPase in inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, mimicked the effect of bradykinin and histamine. In the fibroblasts pretreated with tyrosine kinase inhibitors, the bradykinin-, histamine- and thapsigargin-induced Ca(2+) entry was clearly enhanced, but not the transient [Ca(2+)](i) increase. Tyrosine phosphatase inhibitor benzylphosphonic acid (200 microM) had no effect on Ca(2+)entry or transient [Ca(2+)](i) increase. These results suggest that tyrosine phosphorylation is involved in Ca(2+) entry in human gingival fibroblasts.     

Effects of UV dose on formation of spontaneously developing pocks in Streptomyces azureus ATCC14921

Spontaneously developing pocks (S pocks) of Streptomyces azureus ATCC14921 were formed by the both functions of conjugative plasmid pSA1 and lysogenic phage SAt2. The formation was affected by the dose of UV irradiation. The mean pock diameter in cultures treated with UV light at 0, 7.1, 14.2 and 21.3 x 10(2) microW. erg/cm, respectively, were 1.3, 0.4, 2.2, and 0.5 mm. The dose affected conjugative plasmid pSA1 related to pock formation. There was UV damage of autonomous pSA1 replicon and UV induction of the chromosomal integrated sequence. Increases and decreases in the amount of autonomous pSA1 replicon corresponded to increases and decreases, respectively, in the diameter of the pocks. Both pSA1 and SAt2 syntheses were developed in the large pocks (1.3 and 2.2 mm), but only SAt2 synthesis was developed in the pinhole pocks (0.4 and 0.5 mm).     

Presence of free D-amino acids in microalgae

Several species of microalgae (phytoplankton), 4 species of freshwater algae and 4 species of marine diatoms, were cultured germ-free in the laboratory. The presence of free D-amino acids was verified in these species by a reversed-phase HPLC analysis. D-Aspartate was detected in all the microalgae examined, but D-alanine was only present in the marine diatoms. The D-amino acid content in Asterionella sp. of the marine diatoms increased from the exponential phase to the stationary phase and then decreased to the phase of decline.     

Expression of endothelial nitric oxide synthase in the porcine oocyte and its possible function

The present study was designed to investigate the localization of endothelial nitric oxide synthase (eNOS) in porcine oocytes and its possible function during in vitro development. RT-PCR and immunoblotting analyses revealed the presence of eNOS in the oocytes prepared from small follicles, with an amplified product of 456 bp and an apparent mol wt of 130 kDa, respectively. The synthesis of oocyte NO was suppressed during a 72-h culture of cumulus-oocyte complexes in the presence of follicle-stimulating hormone (FSH), but not luteinizing hormone (LH). However, the decrease in NO synthesis did not result from the levels of eNOS mRNA and its protein, as revealed by analyses of RT-PCR and Western blot analysis, suggesting that expression of oocyte eNOS is not dependent upon gonadotropin stimulation. In proliferated cumulus cells, LH receptor mRNA expression was detected after a 48-h culture with FSH, as revealed by RT-PCR analysis. mRNA expression was inhibited by an NO-releasing agent (S-nitroso-N-acetyl-DL-penicillamine) after an additional 24-h culture. These results suggest that oocytes may release eNOS-derived NO as a signal for somatic cells to steadily suppress the development of cumulus cells, if not FSH stimulation. Conversely, the synthesis of NO is suppressed during the action of FSH on the cumulus cells with no changes in eNOS expression.