HCO(3)(-)-independent rescue from apoptosis by stilbene derivatives in rat cardiomyocytes

Apoptosis of rat cardiomyocytes induced by staurosporine is prevented by a stilbene derivative (DIDS), which is a known blocker of both Cl(-)/HCO(3)(-) exchangers and Cl(-) channels. To clarify its target, staurosporine-induced activation of caspase-3, DNA laddering and cell death were examined in cultured rat cardiomyocytes. Removal of ambient HCO(3)(-), which minimizes the function of Cl(-)/HCO(3)(-) exchangers, failed to affect the preventive effect of DIDS on apoptosis. A carboxylate analog Cl(-) channel blocker, which does not block Cl(-)/HCO(3)(-) exchangers, also inhibited apoptotic events. Thus, rescue by DIDS of cardiomyocytes from apoptosis is mediated by blockage of Cl(-) channels.     

A novel role of CD30/CD30 ligand signaling in the generation of long-lived memory CD8+ T cells

Memory CD8+ T cells can be divided into two subsets, central memory (T(CM)) and effector memory (T(EM)) CD8+ T cells. We found that CD30, a member of the TNFR-associated factor (TRAF)-linked TNFR superfamily, signaling is involved in differentiation of long-lived CD8+ T(CM) cells following Listeria monocytogenes infection. Although CD8+ T(EM) cells transiently accumulated in the nonlymphoid tissues of CD30 ligand (CD153-/-) mice after infection, long-lived memory CD8+ T(CM) cells were poorly generated in these mice. CCR7 mRNA expression was down-regulated in CD8+ T cells of the spleen of CD153-/- mice in vivo and the expression was up-regulated in CD8+ T(EM) cells by anti-CD30 mAb cross-linking in vitro. These results suggest that CD30/CD30 ligand signaling plays an important role in the generation of long-lived memory CD8+ T cells at least partly by triggering homing receptors for T(CM) cells.     

Genetic relationships among Brazilian strains of Aspergillus ochraceus based on RAPD and ITS sequences

Ochratoxin A (OA) is a mycotoxin that has been found in coffee beans and coffee beverages. Its toxicological profile includes carcinogenicity, nephrotoxicity, and immunotoxicity. Aspergillus ochraceus is the major species responsible for OA production in Brazilian coffee beans. The genetic relationships among 25 A. ochraceus strains collected from Brazilian coffee-bean samples were determined based on RAPD and internal transcribed spacer (ITS) sequence data. The isolates were resolved into 2 distinct groups, one with 4 strains (group A) and the other with 21 strains (group B). Specific nucleotide variations characterizing group A and B were found for both ITS1 and ITS2 regions. Group B is a new group proposed here to accommodate the majority of the Brazilian isolates. Each group was found to contain both toxigenic and nontoxigenic strains, indicating that there is no association between molecular genotypes and the ability to produce OA.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

Pigment cells trigger the onset of gastrulation in tropical sea urchin Echinometra mathaei

In the tropical sea urchin Echinometra mathaei, pigment cells are just detectable before the onset of gastrulation, owing to an early accumulation of red pigment granules. Taking advantage of this feature, behavior of pigment cells was studied in relation to the processes of gastrulation. Before the initiation of primary invagination, pigment cells were arranged in a hemi-circle in the dorsal half of the vegetal plate. Inward bending of the vegetal plate first occurred at the position occupied by pigment cells, while the bending was not conspicuous in the ventral half of the blastopore. Rhodamine-phalloidin staining showed that actin filaments were abundant at the apical corticies of pigment cells. It was also found that the onset of gastrulation was considerably delayed in the NiCl2-treated embryos, in which pigment cells were drastically reduced in number. It is notable that the NiCl2-treated embryos began to gastrulate on schedule if they contained a number of pigment cells in spite of treatment. This shows that pigment cells are the bottle cells that trigger the onset of gastrulation. In the embryos devoid of pigment cells, a short stub-like gut rudiment formed in a delayed fashion, and several secondary mesenchyme cells (SMC) appeared at the tip of the rudiment and elongated gradually until its tip reached the apical plate. This observation suggests that the SMC that pull the gut rudiment upward are not pigment cells but blastocoelar cells, because pigment cells change their fate to blastocoelar cells upon NiCl2-treatment.     

Crystal structures and magnetic properties of tetranuclear copper(II) complexes of N-(2-hydroxymethylphenyl)salicylideneimine with a defective double-cubane core

Tetranuclear Cu(II) complexes of N-(2-hydroxymethylphenyl)salicylideneimine (H₂L1-H) and its homologues (5-CH₃: H₂L1-Me, 5-Cl: H₂L1-Cl), [Cu(L1-H)]₄ · 3H₂O (1), [Cu(L1-Me)]₄ · 2CH₂Cl₂ (2), and [Cu(L1-Cl)]₄ · 2CH₂Cl₂ (3), have been characterized by X-ray crystal structure analyses and magnetic measurements. The structure analyses revealed that the complexes 1-3 have a defective double-cubane tetra copper(II) core connected by μ₃-alkoxo bridges. The intramolecular Cu?Cu distances are in the range from 5.251(2)-5.256(3) Å for the longest to 3.0518(9)-3.092(2) Å for the shortest. Each Cu(II) ion has a square-pyramidal geometry and the dihedral angles between adjacent Cu(II) basal planes are almost right angles. Magnetic measurements of the present complexes indicate that weak antiferromagnetic interactions (J=−15 to −19 cm⁻¹) between neighboring copper(II) ions are dominant in these tetracopper cores.     

GAT (GGA and Tom1) domain responsible for ubiquitin binding and ubiquitination

GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins) are a family of monomeric adaptor proteins involved in membrane trafficking from the trans-Golgi network to endosomes. The GAT (GGA and Tom1) domains of GGAs have previously been shown to interact with GTP-bound ARF and to be crucial for membrane recruitment of GGAs. Here we show that the C-terminal subdomain of the GAT domain, which is distinct from the N-terminal GAT subdomain responsible for ARF binding, can bind ubiquitin. The binding is mediated by interactions between residues on one side of the alpha3 helix of the GAT domain and those on the so-called Ile-44 surface patch of ubiquitin. The binding of the GAT domain to ubiquitin can be enhanced by the presence of a GTP-bound form of ARF. Furthermore, GGA itself is ubiquitinated in a manner dependent on the GAT-ubiquitin interaction. These results delineate the molecular basis for the interaction between ubiquitin and GAT and suggest that GGA-mediated trafficking is regulated by the ubiquitin system as endosomal trafficking mediated by other ubiquitin-binding proteins.     

Two extracellular proteins with alkaline peroxidase activity, a novel cytochrome c and a catalase-peroxidase, from Bacillus sp. No.13

A novel cytochrome c and a catalase-peroxidase with alkaline peroxidase activity were purified from the culture supernatant of Bacillus sp. No.13 and characterized. The cytochrome c exhibited absorption maxima at 408 nm (Soret band) in its oxidized state, and 550 (alpha-band), 521 (beta-band), and 415 (Soret band) nm in its reduced state. The native cytochrome c with a relative molecular mass of 15,000 was composed of two identical subunits. The cytochrome c showed over 50 times higher peroxidase activity than those of known c-type cytochromes from various sources. The optimum pH and temperature of the peroxidase activity were about 10.0 and 70 degrees C, respectively. The peroxidase activity is stable in the pH range of 6.0 to 10.8 (30 degrees C, 1-h treatment), and at temperatures up to 80 degrees C (pH 8.5, 20-min treatment). The heme content was determined to be 1 heme per subunit. The amino acid sequence of the cytochrome c showed high homology with those of the c-type cytochromes from Bacillus subtilis and Bacillus sp. PS3. The catalase-peroxidase showed high catalase activity and considerable peroxidase activity, the specific activities being 55,000 and 0.94 micromol/min/mg, respectively. The optimum pH and temperature of the peroxidase activity were in the range of 6.4 to 10.1 and 60 degrees C, respectively. The catalase-peroxidase showed a lower K(m) value (0.67 mM) as to H(2)O(2) than known catalase-peroxidases.     

Acute effect of corticosterone on N-methyl-D-aspartate receptor-mediated Ca2+ elevation in mouse hippocampal slices

We examined the rapid effects of corticosterone (CORT) on N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ signals in adult mouse hippocampal slices by using Ca2+ imaging technique. Application of NMDA caused a transient elevation of intracellular Ca2+ concentration followed by a decay to a plateau within 150s. The 30min preincubation of CORT induced a significant decrease of the peak amplitude of NMDA-induced Ca2+ elevation in the CA1 region. The rapid effect of CORT was induced at a stress-induced level (0.4-10microM). Because the membrane non-permeable bovine serum albumin-conjugated CORT also induced a similar rapid effect, the rapid effect of CORT might be induced via putative surface CORT receptors. In contrast, CORT induced no significant effects on NMDA-induced Ca2+ elevation in the dentate gyrus. In the CA3 region, CORT effects were not evaluated, because the marked elevation of NMDA-induced Ca2+ signals was not observed there.