PAL31, a nuclear protein required for progression to the S phase

PAL31 is a nuclear protein expressed by various cell types. In the present study, the expression and function of PAL31 were examined in the cytokine-regulated growth of T and B cell lines. Treatment of the cells with mitogens [ovine PRL, recombinant rat placental lactogen-I (PL-I) and human IL-3] caused a dose-dependent increase in the expression of PAL31 mRNA in the PRL-dependent cell line Nb2, and IL-3 dependent cell line BaF3. A time-course study on synchronized Nb2 cells revealed that the expression of PAL31 is specific to the late G1 and S phases. Immunocytological studies revealed that PAL31 accumulates in the nuclei at the S phase. Furthermore, the antisense oligonucleotide for PAL31 severely inhibited the proliferation of Nb2 cells by inhibiting cells progressing to the S phase. Thus, PAL31 is a nuclear protein associated with cell cycle progression.     

Amyloid beta proteins inhibit Cl(-)-ATPase activity in cultured rat hippocampal neurons

Cl(-)-ATPase in the CNS is a candidate for an outwardly directed neuronal Cl(-) transporter requiring phosphatidylinositol-4-phosphate (PI4P) for its optimal activity. To test its pathophysiological changes in a phosphatidylinositol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheimer's disease (AD), amyloid beta proteins (Abetas), on the Cl(-)-ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid beta proteins (1-40, 1-42 and 25-35) concentration-dependently (1-100 nM) and time-dependently (from 1 h to 6 day) decreased Cl(-)-ATPase activity and elevated intracellular Cl(-) concentrations ([Cl(-)]i), Abeta25-35 being the most potent. Addition of inositol or 8-Br-cyclic GMP completely reversed these Abeta-induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4-kinase, 10 microM wortmannin or 20 microM quercetin, but not by a PI 3-kinase inhibitor, 50 nM wortmannin or 10 microM LY294002. The PI, PIP and PIP2 levels of the plasma membrane-rich fraction were lower in the Abeta-treated cells as compared with each control. In the Abeta-exposed culture, but not in control, stimulation by 10 microM glutamate for 10 min significantly increased fragmentation of DNA and decreased cell viability. Addition of inositol or 8-Br-cyclic GMP prevented the effect of Abeta-treatment on the neurotoxicity of glutamate. Thus, Abetas reduce neuronal Cl(-)-ATPase activity, resulting in an increase in [Cl(-)]i probably by lowering PI4P levels, and this may reflect a pre-apoptotic condition in early pathophysiological profiles of AD.     

Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments.     

Improvement of the physical properties of pepsin-solubilized elastin-collagen film by crosslinking

Pepsin-solubilized elastin (PSE)-conjugated collagen film was prepared from a collagen matrix with PSE by drying it and crosslinking the constituents with water-soluble carbodiimide or microbial transglutaminase to improve the physical properties of the collagen film. The crosslinking reduced the solubility and improved the thermal stability, the thermal transition properties, and the elasticity of the control film in water. In particular, water-soluble carbodiimide strongly influenced these properties. The PSE-conjugated collagen film showed good permeation by water-soluble tasting substances such as oligosaccharides and amino acids, but poor permeation by polysaccharide, protein, and hydrophobic substances such as retinol and cholesterol.     

An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin

beta-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of beta-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. beta-catenin levels are regulated by the ubiquitin-dependent proteolysis system and beta-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen synthase kinase-3beta (GSK-3beta)/Axin kinase complex. Here we show that FWD1 (the mouse homologue of Slimb/betaTrCP), an F-box/WD40-repeat protein, specifically formed a multi-molecular complex with beta-catenin, Axin, GSK-3beta and APC. Mutations at the signal-induced phosphorylation site of beta-catenin inhibited its association with FWD1. FWD1 facilitated ubiquitination and promoted degradation of beta-catenin, resulting in reduced cytoplasmic beta-catenin levels. In contrast, a dominant-negative mutant form of FWD1 inhibited the ubiquitination process and stabilized beta-catenin. These results suggest that the Skp1/Cullin/F-box protein FWD1 (SCFFWD1)-ubiquitin ligase complex is involved in beta-catenin ubiquitination and that FWD1 serves as an intracellular receptor for phosphorylated beta-catenin. FWD1 also links the phosphorylation machinery to the ubiquitin-proteasome pathway to ensure prompt and efficient proteolysis of beta-catenin in response to external signals. SCFFWD1 may be critical for tumor development and suppression through regulation of beta-catenin protein stability.     

Ecto-ATPase activity in the rat cardiac muscle: biochemical characteristics and histocytochemical localization

We used a combined biochemical and histocytochemical approach to study ecto-ATPase in the rat cardiac muscle. The reaction medium employed for histocytochemical detection was optimized in biochemical assays to achieve the highest enzyme activity and lowest inhibition by the capture agent used for visualization of the reaction product. Approximately 70% of the enzyme activity was retained in samples after the fixation procedure. Divalent cations stimulated ecto-ATPase. High activity was detectable within a wide pH range. Histocytochemical reaction was observed at sites at which extracellular ATP can potentially exert its actions on the cardiac muscle: nerve endings, plasma membranes of cardiac myocytes and capillary endothelial cells, and T-tubules. Product of the reaction was found exclusively at the outer surface of the cells. In controls, enzyme activity was abolished by diethyl pyrocarbonate and slightly stimulated by digitonin and concanavalin A, whereas sodium orthovanadate, N-ethylmaleimide, and sodium azide yielded no effect. Our results support the view that cardiac ecto-ATPase is involved in important physiological functions and suggest that its activity may be regulated by the release of ATP from nerve endings.     

Regulatory molecules for coronary expressions of VEGF and its angiogenic receptor KDR in hypoestrogenic middle-aged female rats

We studied the effects of estrogen deprivation and replacement on the protein and gene expression levels molecules that can be considered to be essential for coronary angiogenesis in middle-aged female rats. The animals were subjected to sham operation, ovariectomy, or ovariectomy with estrogen replacement therapy (ERT). Following ovariectomy, protein and gene expressions of vascular endothelial growth factor (VEGF) and its angiogenic receptor (KDR) showed a marked decline in coronary vessels, as determined by immunohistochemistry and in situ hybridization. ERT resulted in restoration of the ovariectomy-induced changes to intact levels. The coronary expression level of basic fibroblast growth factor was unaffected by estrogen deprivation or treatment. The changes in VEGF and KDR expressions were strongly associated with those in endothelial nitric oxide synthase (eNOS) expression in coronary vessels. Moreover, the age- and gender-dependent accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) protein appeared to be a determinant molecule of VEGF expression in middle-aged female rats. We reached a conclusion that the VEGF-KDR system plays a key role in coronary angiogenesis in hypoestrogenic elderly women and is critically regulated by estrogen, eNOS and HIF-1alpha.     

Estrogen regulation of nitric oxide synthesis in the porcine oocyte

The endothelial type (NOS-3) of three isoforms of nitric oxide (NO) synthase occurs in porcine oocytes and granulosa cells, but the regulation of NO synthesis in oocytes remains unknown. The present study was designed to evaluate steroid control in the process of oocyte NO synthesis. Cumulus-oocyte complexes (COCs), obtained from small-sized antral follicles of immature porcine ovaries, were cultured in estrogen-deprived medium, and the effect of steroids or steroid-free porcine follicular fluids on the NO release from oocytes was investigated. Oocytes that were isolated from cultured COCs were incubated with 1 microM ionomycin. The NO metabolites were identified using a NO detector-high-pressure liquid chromatography system. Oocytes from COCs cultured with 10 nM 17beta-estradiol (E2) released NO in response to ionomycin, whereas progesterone and testosterone had little effect on the synthesis of NO. An inhibitor of NOS suppressed the synthesis of NO. The maximal synthesis was observed after a 15 h-culture with E2. However, oocytes freshly obtained from antral follicles did not response to ionomycin, and the E2 action was suppressed by the addition of steroid-free follicular fluids. Analyses of RT-PCR and Western blotting showed that E2 did not increase NOS-3 expression. In addition, estrogen receptor beta was detected in oocytes and cumulus cells, and estrogen receptor alpha was detected only in cumulus cells. These findings suggest that oocyte NOS-3 is promoted for the synthesis of NO by E2 without increases in NOS-3 expression, but the synthesis of NO is suppressed, at least in the oocytes of early antral follicles.     

Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism

Symbiobacterium thermophilum is an uncultivable bacterium isolated from compost that depends on microbial commensalism. The 16S ribosomal DNA-based phylogeny suggests that this bacterium belongs to an unknown taxon in the Gram-positive bacterial cluster. Here, we describe the 3.57 Mb genome sequence of S.thermophilum. The genome consists of 3338 protein-coding sequences, out of which 2082 have functional assignments. Despite the high G + C content (68.7%), the genome is closest to that of Firmicutes, a phylum consisting of low G + C Gram-positive bacteria. This provides evidence for the presence of an undefined category in the Gram-positive bacterial group. The presence of both spo and related genes and microscopic observation indicate that S.thermophilum is the first high G + C organism that forms endospores. The S.thermophilum genome is also characterized by the widespread insertion of class C group II introns, which are oriented in the same direction as chromosomal replication. The genome has many membrane transporters, a number of which are involved in the uptake of peptides and amino acids. The genes involved in primary metabolism are largely identified, except those that code several biosynthetic enzymes and carbonic anhydrase. The organism also has a variety of respiratory systems including Nap nitrate reductase, which has been found only in Gram-negative bacteria. Overall, these features suggest that S.thermophilum is adaptable to and thus lives in various environments, such that its growth requirement could be a substance or a physiological condition that is generally available in the natural environment rather than a highly specific substance that is present only in a limited niche. The genomic information from S.thermophilum offers new insights into microbial diversity and evolutionary sciences, and provides a framework for characterizing the molecular basis underlying microbial commensalism.     

Formation mechanism of 2,6-dimethyl-2,6-octadienes from thermal decomposition of linalyl beta-D-glucopyranoside

Thermally decomposed products of (+/-)-linalyl beta-D-glucoside were analyzed by GC and GC/MS. 2,6-dimethyl-2,6-octadienes produced by mild pyrolysis of linalyl beta-D-glucopyranoside under a vacuum were detected and characterized by MS and NMR spectroscopy. This suggests that 2,6-dimethyl-2,6-octadienes are produced during thermal decomposition of the glucoside via proton transfer from the anomeric position to C-6 in the aglycon moiety. A stable isotope labeling experiment directly indicated the new reaction mechanism.