CD9 is associated with leukemia inhibitory factor-mediated maintenance of embryonic stem cells

Mouse embryonic stem (ES) cells can proliferate indefinitely in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), or differentiate into all three germ layers upon removal of this factor. To determine cellular factors associated with self-renewal of undifferentiated ES cells, we used polymerase chain reaction-assisted cDNA subtraction to screen genes that are expressed in undifferentiated ES cells and down-regulated after incubating these cells in a differentiation medium without LIF for 48 h. The mRNA expression of a tetraspanin transmembrane protein, CD9, was high in undifferentiated ES cells and decreased shortly after cell differentiation. An immunohistochemical analysis confirmed that plasma membrane-associated CD9 was expressed in undifferentiated ES cells but low in the differentiated cells. Addition of LIF to differentiating ES cells reinduced mRNA expression of CD9, and CD9 expression was accompanied with a reappearance of undifferentiated ES cells. Furthermore, activation of STAT3 induced the expression of CD9, indicating the LIF/STAT3 pathway is critical for maintaining CD9 expression. Finally, addition of anti-CD9 antibody blocked ES cell colony formation and reduced cell viability. These results indicate that CD9 may play a role in LIF-mediated maintenance of undifferentiated ES cells.     

Proteomic characterization and functional analysis of outer membrane vesicles of Francisella novicida suggests possible role in virulence and use as a vaccine

We have isolated and characterized outer membrane vesicles (OMVs) from Francisella. Transport of effector molecules through secretion systems is a major mechanism by which Francisella tularensis alters the extracellular proteome and interacts with the host during infection. Outer membrane vesicles produced by Francisella were examined using TEM and AFM and found to be 43-125 nm in size, representing another potential mechanism for altering the extracellular environment. A proteomic analysis (LC-MS/MS) of OMVs from F. novicida and F. philomiragia identified 416 (F. novicida) and 238 (F. philomiragia) different proteins, demonstrating that OMVs are an important contributor to the extracellular proteome. Many of the identified OMV proteins have a demonstrated role in Francisella pathogenesis. Biochemical assays demonstrated that Francisella OMVs possess acid phosphatase and hemolytic activities that may affect host cells during infection, and are cytotoxic toward murine macrophages in cell culture. OMVs have been previously used as a human vaccine against Neisseria meningitidis . We hypothesized that Francisella OMVs could be useful as a novel Francisella vaccine. Vaccinated BALB/C mice challenged with up to 50 LD50 of Francisella showed statistically significant protection when compared to control mice. In the context of these new findings, we discuss the relevance of OMVs in Francisella pathogenesis as well as their potential use as a vaccine.     

Characterization of the stromal protease(s) degrading the cross-linked products of the D1 protein generated by photoinhibition of photosystem II

When photosystem (PS) II-enriched membranes are exposed to strong light, cross-linking of the intrinsic D1 protein with the surrounding polypeptides and degradation of the D1 protein take place. The cross-linking of the D1 protein with the alpha-subunit of cytochrome b(559) is suggested to be an early event of photoinduced damage to the D1 protein (Barbato et al., FEBS Lett. 309 (1992) 165-169). The relationship between the cross-linking and the degradation of the D1 protein, however, is not yet clear. In the present study, we show that the addition of stromal extract from chloroplasts degrades the 41 kDa cross-linked product of D1/cytochrome b(559) alpha-subunit and enhances the degradation of the D1 protein. Incubation of the preilluminated PS II-enriched membranes with the stromal extract at 25 degrees C causes the degradation of the cross-linked product by more than 70%. The activity of the stromal extract showed a pH optimum at 8.0, and was enhanced by the addition of ATP or GTP. Consistent with the nucleotide effect, this stromal activity was eliminated by the preincubation of the stromal extract with apyrase, which hydrolyzes nucleotides. Also, the stromal activity was nearly fully inhibited by a serine-type protease inhibitor, 3,4-dichloroisocoumarin, which suggests participation of a serine-type protease(s).     

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15–20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a λ phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis. Received: April 17, 1998 / Accepted: June 23, 1998     

Photoperiodic response of serotonin- and galanin-immunoreactive neurons of the paraventricular organ and infundibular nucleus in Japanese quail, Coturnix coturnix japonica

We investigated the photoperiodic response of serotonin- and galanin (GA)- immunoreactive (ir) cells in the paraventricular organ (PVO) and infundibular nucleus (IF) of the Japanese quail and the interaction of these cells with gonadotropin-releasing hormone (GnRH)-ir neurons in the hypothalamus. Serotonin-ir cells were located in series from the PVO to the IF, and were connected with each other. The number of serotonin-ir cells differed significantly between light and dark phases on the short days (SD), but did not differ between light and dark phases on long days (LD). GA-ir cells were also found in the PVO and IF. The number of GA-ir cells under SD conditions was significantly greater than under LD conditions but did not change diurnally. Both serotonin-ir and GA-ir fibers ran along the GnRH-ir cells in the nucleus commissurae pallii. Serotonin-ir and GA-ir fibers were connected with the GnRH-ir fibers in the external layer of the median eminence (ME). We confirmed that GA-ir fibers were closely associated with serotonin-ir neurons in the PVO and IF. GA-ir neurons have at least 2 routes of regulating GnRH neurons directly, and indirectly via the serotonin-ir cells in the PVO and IF.     

Synthesis and biological activity of novel alpha-substituted beta-phenylpropionic acids having pyridin-2-ylphenyl moiety as antihyperglycemic agents

We previously reported the identification of novel oximes having 5-benzyl-2,4-thiazolidinedione with antihyperglycemic activity. We now report the synthesis and biological activity of a novel series of oximes and amides having alpha-substituted-beta-phenylpropionic acids. In this series, we obtained potent PPAR alpha/gamma dual agonist (S)-9d, with which activation of PPAR alpha and PPAR gamma was considerably more potent than that of the reference compounds GW9578 22 and rosiglitazone 3, respectively. This means (S)-9d is of the strongest class of PPAR alpha/gamma dual agonists. In the course of this study, we also obtained 8h, which indicated potent plasma glucose lowering effect in spite of weak PPAR alpha/gamma agonistic activity.     

Expression of programmed death 1 ligands by murine T cells and APC

Programmed death 1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral tolerance. Two ligands for PD-1, namely, B7-H1 (PD-L1) and B7-DC (PD-L2), have recently been identified as new members of the B7 family but their expression at the protein level remains largely unknown. To characterize the expression of B7-H1 and B7-DC, we newly generated an anti-mouse B7-H1 mAb (MIH6) and an anti-mouse B7-DC mAb (TY25). MIH6 and TY25 immunoprecipitated a single molecule of 43 and 42 kDa from the lysate of B7-H1 and B7-DC transfectants, respectively. Flow cytometric analysis revealed that B7-H1 was broadly expressed on the surface of mouse tumor cell lines while the expression of B7-DC was rather restricted. PD-1 was expressed on anti-CD3-stimulated T cells and anti-IgM plus anti-CD40-stimulated B cells at high levels but was undetectable on activated macrophages or DCs. B7-H1 was constitutively expressed on freshly isolated splenic T cells, B cells, macrophages, and dendritic cells (DCs), and up-regulated on T cells by anti-CD3 stimulation on macrophages by LPS, IFN-gamma, GM-CSF, or IL-4, and on DCs by IFN-gamma, GM-CSF, or IL-4. In contrast, B7-DC expression was only inducible on macrophages and DCs upon stimulation with IFN-gamma, GM-CSF, or IL-4. The inducible expression of PD-1 ligands on both T cells and APCs may suggest new paradigms of PD-1-mediated immune regulation.