Interaction between a negative regulator (Msa2/Nrd1) and a positive regulator (Cpc2) of sexual differentiation in Schizosaccharomyces pombe

The sexual differentiation of Schizosaccharomyces pombe is controlled by many cellular components which have not been fully characterized. We isolated a gene called msa2 as a multi-copy suppressor of a sporulation abnormal mutant (sam1). Msa2p is identical with Nrd1p which has been characterized as a factor that blocks the onset of sexual differentiation. The yeast two-hybrid system was used to identify Cpc2p, a fission yeast homolog of the RACK1 protein, that interacted with Msa2p/Nrd1p. We confirmed that Msa2p/Nrd1p interacted with Cpc2p in S. pombe cells. An epistatic analysis of msa2/nrd1 and cpc2 suggests that Msa2p/Nrd1p was an upstream regulator for Cpc2p. A localization analysis of Cpc2p and Msa2p/Nrd1p indicates that both proteins were predominantly localized in the cytoplasm. The interaction of negative regulator Msa2p/Nrd1p with positive regulator Cpc2p suggests a new regulatory circuit in the sexual differentiation of S. pombe.     

Development of definitive endoderm from embryonic stem cells in culture

The cellular and molecular events regulating the induction and tissue-specific differentiation of endoderm are central to our understanding of the development and function of many organ systems. To define and characterize key components in this process, we have investigated the potential of embryonic stem (ES) cells to generate endoderm following their differentiation to embryoid bodies (EBs) in culture. We found that endoderm can be induced in EBs, either by limited exposure to serum or by culturing in the presence of activin A (activin) under serum-free conditions. By using an ES cell line with the green fluorescent protein (GFP) cDNA targeted to the brachyury locus, we demonstrate that endoderm develops from a brachyury(+) population that also displays mesoderm potential. Transplantation of cells generated from activin-induced brachyury(+) cells to the kidney capsule of recipient mice resulted in the development of endoderm-derived structures. These findings demonstrate that ES cells can generate endoderm in culture and, as such, establish this differentiation system as a unique murine model for studying the development and specification of this germ layer.     

Inhibition of sphingomyelinase activity helps to prevent neuron death caused by ischemic stress

Magnesium-dependent neutral sphingomyelinase (N-SMase) present in plasma membranes is an enzyme that can be activated by stress in the form of inflammatory cytokines, serum deprivation, and hypoxia. The design of small molecule N-SMase inhibitors may offer new therapies for the treatment of inflammation, ischemic injury, and cerebral infarction. Recently, we synthesized a series of difluoromethylene analogues (SMAs) of sphingomyelin. We report here the effects of SMAs on the serum/glucose deprivation-induced death of neuronally differentiated pheochromocytoma (PC-12) cells and on cerebral infarction in mice. SMAs inhibited the enhanced N-SMase activity in the serum/glucose-deprived PC-12 cells, and thereby suppressed the apoptotic sequence: ceramide formation, c-Jun N-terminal kinase phosphorylation, caspase-3 activation, and DNA fragmentation in the nuclei. Administration of SMA-7 (10 mg/kg i.v.) with IC50= 3.3 microM to mice whose middle cerebral arteries were occluded reduced significantly the size of the cerebral infarcts, compared to the control mice. These results suggest that N-SMase is a key component of the signaling pathways in cytokine- and other stress-induced cellular responses, and that inhibiting or stopping N-SMase activity is an important strategy to prevent neuron death from ischemia.     

Instream distributions and feeding habits of two species of sleeper, <Emphasis Type="Italic">Eleotris acanthopoma</Emphasis> and <Emphasis Type="Italic">Eleotris fusca</Emphasis>, in the Teima River,Okinawa Island

The instream distributions and feeding habits of two species of sleeper, Eleotris acanthopoma and E. fusca, were studied in the Teima River on Okinawa Island, southern Japan. Both adult fishes inhabited the river, but their distribution patterns were found to be different. The distribution of E. acanthopoma was from the tidally influenced area to the lower part of the freshwater area, whereas E. fusca was distributed almost entirely in the freshwater area. They were found to coexist at the upper limit of the tidally influenced area and the lower part of the freshwater area. Their feeding habits were clearly different, although both species were carnivorous. Eleotris acanthopoma fed mainly on crabs in the tidally influenced area and on aquatic snails in the freshwater area, where they coexist with E. fusca. In contrast, E. fusca fed mainly on shrimps in the freshwater area. Their coexistence may result from the difference in their feeding habits.     

A new hypothesis for species coexistence: male–male repulsion promotes coexistence of competing species

We propose a new hypothesis for species coexistence by considering behavioral interactions between individuals. The hypothesis states that repulsive behavior between conspecific males (male–male repulsion) creates space for competing species, which promotes their coexistence. This hypothesis can explain the coexistence of two competing species even when their ecological niches completely overlap in spatially homogeneous environments. In addition, the mechanisms underlying such behavior might play a role in enabling the coexistence of two species immediately after speciation, with little or no niche differentiation, as in the case of cichlid fish communities, for example. Although there is limited evidence supporting this hypothesis, it can nevertheless explain the occurrence of species coexistence and biodiversity, which cannot be explained by previous theories.     

The ATM-related Tel1 protein of Saccharomyces cerevisiae controls a checkpoint response following phleomycin treatment

MEC1 and TEL1 encode ATR- and ATM-related proteins in the budding yeast Saccharomyces cerevisiae, respectively. Phleomycin is an agent that catalyzes double-strand breaks in DNA. We show here that both Mec1 and Tel1 regulate the checkpoint response following phleomycin treatment. MEC1 is required for Rad53 phosphorylation and cell-cycle progression delay following phleomycin treatment in G1, S or G2/M phases. The tel1Δ mutation confers a defect in the checkpoint responses to phleomycin treatment in S phase. In addition, the tel1Δ mutation enhances the mec1 defect in activation of the phleomycin-induced checkpoint pathway in S phase. In contrast, the tel1Δ mutation confers only a minor defect in the checkpoint responses in G1 phase and no apparent defect in G2/M phase. Methyl methanesulfonate (MMS) treatment also activates checkpoints, inducing Rad53 phosphorylation in S phase. MMS-induced Rad53 phosphorylation is not detected in mec1Δ mutants during S phase, but occurs in tel1Δ mutants similar to wild-type cells. Finally, Xrs2 is phosphorylated after phleomycin treatment in a TEL1-dependent manner during S phase, whereas no significant Xrs2 phosphorylation is detected after MMS treatment. Together, our results support a model in which Tel1 contributes to checkpoint control in response to phleomycin-induced DNA damage in S phase.     

Effects of donor cell type and genotype on the efficiency of mouse somatic cell cloning

Although it is widely assumed that the cell type and genotype of the donor cell affect the efficiency of somatic cell cloning, little systematic analysis has been done to verify this assumption. The present study was undertaken to examine whether donor cell type, donor genotype, or a combination thereof increased the efficiency of mouse cloning. Initially we assessed the developmental ability of embryos that were cloned from cumulus or immature Sertoli cells with six different genotypes (i.e., 2 x 6 factorial). Significantly better cleavage rates were obtained with cumulus cells than with Sertoli cells (P < 0.005, two-way ANOVA), which probably was due to the superior cell-cycle synchrony of cumulus cells at G0/G1. After embryo transfer, there was a significant effect of cell type on the birth rate, with Sertoli cells giving the better result (P < 0.005). Furthermore, there was a significant interaction (P < 0.05) between the cell type and genotype, which indicates that cloning efficiency is determined by a combination of these two factors. The highest mean birth rate (10.8 +/- 2.1%) was obtained with (B6 x 129)F1 Sertoli cells. In the second series of experiments, we examined whether the developmental ability of clones with the wild-type genotype (JF1) was improved when combined with the 129 genotype. Normal pups were cloned from cumulus and immature Sertoli cells of the (129 x JF1)F1 and (JF1 x 129)F1 genotypes, whereas no pups were born from cells with the (B6 x JF1)F1 genotype. The present study clearly demonstrates that the efficiency of somatic cell cloning, and in particular fetal survival after embryo transfer, may be improved significantly by choosing the appropriate combinations of cell type and genotype.     

Enhanced chemiluminescence of 6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one by attachment of a cyclomaltooligosaccharide (cyclodextrin). Attachment of cyclomaltononaose (delta-cyclodextrin)

2-Methyl-6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one (MCLA) is an oxygen-induced chemiluminescent compound. It has been shown that the chemiluminescence can be enhanced by forming a cyclomaltooligosaccharide (cyclodextrin)-bound MCLA, and therefore, in continuation of the survey of the types of cyclodextrins, in this study, MCLA was attached to the secondary hydroxyl face of delta-cyclodextrin, which consists of nine D-glucose units. Although the oxygen-induced chemiluminescence efficiency of delta-cyclodextrin-bound MCLA in a pH 8.0 aqueous phosphate buffer was 12 times greater than that of MCLA, the efficiency was markedly lower than that of gamma-cyclodextrin-bound MCLA, which exhibited the highest chemiluminescence efficiency in the previous investigation. Although fluorescence efficiency and light-emitter formation efficiency for delta-cyclodextrin-bound MCLA were similar to those for gamma-cyclodextrin-bound MCLA, singlet-excited state formation efficiency for delta-cyclodextrin-bound MCLA was lower than that for gamma-cyclodextrin-bound MCLA. This study distinctly indicated the optimum cyclodextrin for construction of greatly luminescent cyclodextrin-bound MCLA is gamma-cyclodextrin.     

Characterization and preliminary crystallographic studies of EMS16, an antagonist of collagen receptor (GPIa/IIa) from the venom of Echis multisquamatus

EMS16 is a member of the snake venom-derived C-type lectin family of proteins (CLPs) found in the venom of Echis multisquamatus. It binds to glycoprotein Ia/IIa (integrin alpha2beta1), a major collagen receptor of platelets, acting as a potent antagonist of platelet aggregation and cell migration. Amino acid sequencing and cDNA cloning of EMS16 have revealed that it is composed of an A chain of 134 amino acid residues and a B chain of 128 residues. Crystals of EMS16 belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.57, b = 59.93, and c = 115.74 A, and diffract to a resolution of 1.9 A. Phase determination is underway by means of molecular replacement with the structure of blood coagulation factor IX-binding protein (IX-bp) from habu snake venom (PDB code 1bj3) as the search model.