Genetic approach and phenotype-based complementation screening for identification of stroma cell-derived proteins involved in cell proliferation.

The functional capacities of stromal cell lines to support stem cell activity are heterogeneous and the mechanism of how they support bone marrow cultures remains unclear. Recently, we reported a strategy of functional analysis in which a genetic approach is combined with phenotype-based complementation screening to search for a novel secreted growth factor from mouse bone marrow stroma called ShIF that supported proliferation of bone marrow cells. To investigate the role of stromal cells in hemopoiesis, we extended this strategy to search for stroma-derived proteins that induce cell proliferation by establishing stroma-dependent Ba/F3 mutants of three stroma cell lines from two mouse tissues. Seven stroma-dependent Ba/F3 mutants were used as responder cells to identify cDNAs from stroma cell lines whose products supported proliferation not only to the mutant cells but also to hemopoietic progenitor cells in vitro.     

Caenorhabditis elegans reticulon interacts with RME-1 during embryogenesis

Reticulon (RTN) family proteins are localized in the endoplasmic reticulum (ER). At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are unknown. Each RTN gene produces 1-3 proteins by different promoters and alternative splicing. In Caenorhabditis elegans, there is a single gene (rtn gene) encoding three reticulon proteins, nRTN-A, B, and C. mRNA of nRTN-C is expressed in germ cells and embryos. However, nRTN-C protein is only expressed during embryogenesis and rapidly disappears after hatch. By yeast two-hybrid screening, two clones encoding the same C-terminal region of RME-1, a protein functioning in the endocytic recycling, were isolated. These findings suggest that nRTN-C functions in the endocytic pathway during embryogenesis.     

Photoreaction of methyl and phenyl 4,6-O-benzylidene-2,3-dideoxy-2-C-p-tolylsulfonyl-beta-D-erythro-hex-2-enopyranosides in methanol

The title compounds were irradiated with a high-pressure mercury lamp in methanol to give 2-C-hydroxymethyl derivatives having the gluco, altro, and allo configurations as well as an S(N)2' product. Equatorial attack of a hydroxymethyl radical slightly predominated over axial attack. During chromatographic separation on a silica gel column, partial migration of the 4,6-O-benzylidene group in the gluco and altro products occurred to yield the 3',4-O-benzylidene derivatives.     

Synthetic sialylphosphatidylethanolamine derivatives bind to human influenza A viruses and inhibit viral infection

We synthesized the sialylphosphatidylethanolamine (sialyl PE) derivatives Neu5Ac-PE, (Neu5Ac)2-PE, Neu5Ac-PE (amide) and Neu5Ac-PE (methyl). We examined the anti-viral effects of the derivatives on human influenza A virus infection by ELISA/virus-binding, hemagglutination inhibition, hemolysis inhibition and neutralization assays. The sialyl PE derivatives that we examined bound to A/Aichi/2/68, A/Singapore/1/57 and A/Memphis/1/71 strains of H3N2 subtype, but not to A/PR/8/34 strain of H1N1 subtype. The derivatives inhibited viral hemagglutination and hemolysis of human erythrocytes with A/Aichi/2/68 and A/Singapore/1/57 (H3N2), but not with A/PR/8/34 (H1N1). The inhibitory activity of the (Neu5Ac)2-PE derivative was the strongest of all sialyl PE derivatives (IC50, 35 M to 40 M). Sialyl PE derivatives also inhibited the infection of A/Aichi/2/68 in MDCK cells. Complete inhibition was observed at a concentration between 0.3 to 1.3 mM. IC50 of (Neu5Ac)2-PE was 15 M in A/Aichi/2/68 strain. Taken together, the synthetic sialyl PE derivatives may be effective reagents against infection of some types of influenza A viruses.     

Inflammatory responses to ischemia,and reperfusion in skeletal muscle

Skeletal muscle ischemia and reperfusion is now recognized as one form of acute inflammation in which activated leukocytes play a key role. Although restoration of flow is essential in alleviating ischemic injury, reperfusion initiates a complex series of reactions which lead to neutrophil accumulation, microvascular barrier disruption, and edema formation. A large body of evidence exists which suggests that leukocyte adhesion to and emigration across postcapillary venules plays a crucial role in the genesis of reperfusion injury in skeletal muscle. Reactive oxygen species generated by xanthine oxidase and other enzymes promote the formation of proinflammatory stimuli, modify the expression of adhesion molecules on the surface of leukocytes and endothelial cells, and reduce the bioavailability of the potent antiadhesive agent nitric oxide. As a consequence of these events, leukocytes begin to form loose adhesive interactions with postcapillary venular endothelium (leukocyte rolling). If the proinflammatory stimulus is sufficient, leukocytes may become firmly adherent (stationary adhesion) to the venular endothelium. Those leukocytes which become firmly adherent may then diapedese into the perivascular space. The emigrated leukocytes induce parenchymal cell injury via a directed release of oxidants and hydrolytic enzymes. In addition, the emigrating leukocytes also exacerbate ischemic injury by disrupting the microvascular barrier during their egress across the vasculature. As a consequence of this increase in microvascular permeability, transcapillary fluid filtration is enhanced and edema results. The resultant increase in interstitial tissue pressure physically compresses the capillaries, thereby preventing microvascular perfusion and thus promoting the development of the no-reflow phenomenon. The purpose of this review is to summarize the available information regarding these mechanisms of skeletal muscle ischemia/reperfusion injury.     

Ischemic preconditioning attenuates apoptotic cell death associated with ischemia/reperfusion

Apoptosis or programmed cell death is a genetically controlled response for cells to commit suicide and is associated with DNA fragmentation or laddering. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca2+ which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine whether ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15, 30 or 60 min of ischemia as well as 15 min of ischemia followed by 30, 60, 90 or 120 min of reperfusion. At the end of each experiment, the heart was processed for the evaluation of apoptosis and DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG® in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from the hearts to 1.8% agarose gel electrophoresis and photographed under UV illumination. The results of our study revealed apoptotic cells only in the 90 and 120 min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation which showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). The presence of apoptotic cells and DNA fragmentation in the myocardium were completely abolished by subjecting the myocardium to repeated short-term ischemia and reperfusion which also reduced the ischemic reperfusion injury as evidenced by better recovery of left ventricular performance in the preconditioned myocardium. The results of this study indicate that reperfusion of ischemic heart, but not ischemia, induces apoptotic cell death and DNA fragmentation which can be inhibited by myocardial adaptation to ischemia.     

The mechanism of IL-5 signal transduction

Cytokines are important regulators ofhematopoiesis. They exert their actions by binding to specificreceptors on the cell surface. Interleukin-5 (IL-5) is a criticalcytokine that regulates the growth, activation, and survival ofeosinophils. Because eosinophils play a seminal role in thepathogenesis of asthma and allergic diseases, an understanding of thesignal transduction mechanism of IL-5 is of paramount importance. TheIL-5 receptor is a heterodimer of - and -subunits. The-subunit is specific, whereas the -subunit is common to IL-3,IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF)receptors and is crucial for signal transduction. It has been shownthat there are two major signaling pathways of IL-5 in eosinophils.IL-5 activates Lyn, Syk, and JAK2 and propagates signals through theRas-MAPK and JAK-STAT pathways. Studies suggest that Lyn, Syk, and JAK2tyrosine kinases and SHP-2 tyrosine phosphatase are important foreosinophil survival. In contrast to their survival-promoting activity,Lyn and JAK2 appear to have no role in eosinophil degranulation orexpression of surface adhesion molecules. Raf-1 kinase, on the otherhand, is critical for eosinophil degranulation and adhesion moleculeexpression. Btk is involved in IL-5 stimulation of B cell function.However, it does not appear to be important for eosinophil function.Thus a clear segregation of signaling molecules based on theirfunctional importance is emerging. This review describes the signaltransduction mechanism of the IL-3/GM-CSF/IL-5 receptor system andcompares and contrasts IL-5 signaling between eosinophils and B cells.

     

Effect of phorbol esters on Ca2+ sensitivity and myosin light-chain phosphorylation in airway smooth muscle

We studied in -escin-permeabilized canine tracheal smoothmuscle (CTSM) the effect of the protein kinase C (PKC) agonist phorbol12,13-dibutyrate (PDBu) on isometric force at a constant submaximalCa²⁺ concentration (i.e., theeffect on Ca²⁺ sensitivity) andregulatory myosin light-chain (rMLC) phosphorylation. PDBuincreased Ca²⁺sensitivity, an increase associated with a concentration-dependent, sustained increase in rMLC phosphorylation. PDBu altered therelationship between rMLC phosphorylation and isometric force such thatthe increase in isometric force was less than that expected for the increase in rMLC phosphorylation observed. The effect of four PKCinhibitors [calphostin C, chelerythrine chloride, apseudosubstrate inhibitor for PKC, PKC peptide-(1931) (PSSI), andstaurosporine] on PDBu-inducedCa²⁺ sensitization as well as theeffect of calphostin C and PSSI on rMLC phosphorylation weredetermined. Whereas none of these compounds prevented or reversed thePDBu-induced increase in Ca²⁺sensitivity, the PDBu-induced increase in rMLC phosphorylation wasinhibited. We conclude that PDBu increases rMLC phosphorylation byactivation of PKC but that the associated PDBu-induced increases inCa²⁺ sensitivity are mediated bymechanisms other than activation of PKC in permeabilized airway smoothmuscle.

     

Promoters of Rice Seed Storage Protein Genes Direct Endosperm-Specific Gene Expression in Transgenic Rice

The promoters of genes encoding rice seed storage proteins (glutelin,prolamin, globulin and albumin) were analyzed for their abilityto direct rß-gIucuronidase (GUS) gene expression intransgenic rice plants. All promoters tested could direct endosperm-specificexpression of the GUS reporter gene irrespective of variableactivities and patterns in the endosperm. (Received February 27, 1998; Accepted May 16, 1998)