Deuterostomic Actin Genes and the Definition of the Chordates: cDNA Cloning and Gene Organization for Cephalochordates and Hemichordates

The evolutionary relationship of muscle and nonmuscle actin isoforms in deuterostomia was studied by the isolation and characterization of two actin genes from the cephalochordate Branchiostoma lanceolatum and two from the hemichordate Saccoglossus kowalevskii The Branchiostoma genes specify a muscle and a nonmuscle actin type, respectively. Together with earlier results on muscle actins from vertebrates and urochordates, a N-terminal sequence signature is defined for chordate muscle actins. These diagnostic amino acid residues separate the chordates from the echinoderms and other metazoa. Although the two Saccoglossus actins characterized so far lack the diagnostic residues, in line with the presumptive phylogenetic position of hemichordates outside the chordates, a definitive conclusion can only be expected once the full complement of actin genes of Saccoglossus is established. Comparison of the intron patterns of the various deuterostomic actin genes shows that intron 330-3, which is present in all vertebrate genes, is conspicuously absent from nonvertebrate genes. The possible origin of this intron is discussed. Received: 4 July 1997 / Accepted: 29 August 1997     

Ganglioside GM1 Activates the Mitogen-Activated Protein Kinase Erk2 and p70 S6 Kinase in U-1242 MG Human Glioma Cells

Abstract: Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U-1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [³H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U-1242 MG cells with GM1 caused activation of the mitogen-activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1-stimulated Erk2 activation and GM1-stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70^s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1-stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet-derived growth factor also activated both Erk2 and p70^s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1-mediated DNA synthesis.     

玉米CMS材料线粒体DNA遗传多型性的研究

选用１１×４＝４４个探针／酶组合,５０个（１０ｍｅｒ）随机序列引物对２５种不同胞质来源的ＣＭＳ玉米,５种正常胞质玉米线粒体ＤＮＡ进行ＲＦＬＰ和ＲＡＰＤ研究。研究结果表明：（１）４５％的探针／酶组合可检测到玉米线粒体ＤＮＡ的多型性,共表现１５种ＲＦＬＰ类型,其中Ｓ组ＣＭＳ材料内有７种,正常胞质材料内有２种;８０％的随机引物可检测到ＲＡＰＤ。（２）基于ＲＦＬＰ资料的聚类分析结果,可将３０种胞质明确地划分为Ｔ、Ｃ、Ｓ、Ｎ４组,其结果与恢复专效性测定结果一致。其中ｐＨＪ２－７－１／ＢａｍＨＩ的ＲＦＬＰ类型可成为利用ＲＦＬＰ技术进行胞质分组的鉴定体系。（３）“双”型胞质线粒体ＤＮＡ常表现Ｓ＋Ｃ胞质的ＲＦＬＰ图谱。     

Cpn60 is exclusively localized into mitochondria of rat liver and embryonic Drosophila cells

Several reports have claimed that the mitochondrial chaperonin cpn60, or a close homolog, is also present in some other subcellular compartments of the eukaryotic cell. Immunoelectron microscopy studies, using a polyclonal serum against cpn60, revealed that the protein is exclusively localized within the mitochondria of rat liver and embryonic Drosophila cells (SL2). Furthermore, no cpn60 immunoreactive material could be found within the nucleus of SL2 cells subjected to a 1 h 37°C heat-shock treatment. In contrast to these findings, immunoelectron microscopy studies, using a cpn60 monoclonal antibody, revealed mitochondrial and extramitochondrial (plasma membrane, nucleus) immunoreactive material in rat liver cells. Surprisingly, the monoclonal antibody also reacted with fixed proteins of the mature red blood cell. The monoclonal antibody, as well as cpn60 polyclonal sera, only recognize mitochondrial cpn60 in Western blots of liver proteins. Furthermore, none of the cpn60 antibodies used in this study recognized blotted proteins from rat red blood cells. Therefore, we suggest that the reported extramitochondrial localization of cpn60 in metazoan cells may be due to cross-reactivity of some of cpn60 antibodies with conformational epitopes also present in distantly related cpn60 protein homologs that are preserved during fixation procedures of the cells. © 1995 Wiley-Liss, Inc.     

Interaction of force transmission and sarcomere assembly at the muscle-tendon junctions of carp (Cyprinus carpio): ultrastructure and distribution of titin (connectin) and α-actinin

At muscle-tendon junctions of red and of white axial muscle fibres of carp, new sarcomeres are found adjacent to existing sarcomeres along the bundles of actin filaments that connect the myofibrils with the junctional sarcolemma. As the filament bundles that transmit force to the junction originate proximal to new sarcomeres, they probably relieve these new sarcomeres from premature loading. In red fibres, these filament bundles are long (up to 20 m) and dense, permitting light-microscopical immunohistochemistry (double reactions: anti-titin or anti--actinin and phalloidin). New sarcomeres have clear I bands; their A band lengths are similar to those of older sarcomeres and the thick filaments lie in register. T tubules are found at the distal side of new sarcomeres but terminal Z lines are absent. The late addition of -actinin suggests that -actinin mainly has a stabilizing role in sarcomere formation. The presence of titin in the terminal fibre protrusions is in agreement with its supposed role in sarcomere formation, viz. the integration of thin and thick filaments. The absence of a terminal Z line from sarcomeres with well-registered A bands suggests that this structure is not essential for the anchorage of connective (titin) filaments.     

Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

缺氧促进热休克蛋白70在肺动脉平滑肌细胞中的表达

缺氧性肺动脉高压中肺动脉结构重组,中膜平滑肌细胞增殖并迁移,但机制不明。本研究观察缺氧对肺动脉平滑肌细胞（ＰＡＳＭＣ）细胞周期、ＤＮＡ合成及细胞增殖的影响,并通过观察缺氧对ＰＡＳＭＣ热休克蛋白７０（ＨＳＰ７０）表达的影响,初步探讨缺氧的作用是如何介导的。结果表明缺氧可直接或协同内皮质－１促进ＰＡＳＭＣ ＤＮＡ合成及细胞增殖,并可增加ＨＳＰ７０在ＰＡＳＭＣ中的表达。     

Reversible inhibition of aggregation-related cohesivity in Dictyostelium discoideum by diffusible metabolites

Evidence presented elsewhere (G.B. Williams, E.M. Elder, and M. Sussman 1984, Dev. Biol. 105, 377-388) indicates that NH3 and certain carboxylic acids including propionate, succinate, and acetate modulate the cAMP relay in Dictyostelium discoideum. The former appears to act as a cAMP accumulation inhibitor, the latter as cAMP release inhibitors. The cohesive properties of aggregation competent cells have been assayed quantitatively in the presence of these modulators. The following results were obtained: (1) At pH 7.5, EDTA-resistant cohesivity was greatly inhibited by NH4C within the concentration range tested (30-3.8 mM). Even at the higher concentrations the effect was not immediate but required ca. 10 min for full expression. At the lower concentrations, the inhibitory level was only slightly reduced but the time for full expression progressively increased. At pH 6.5, the level of inhibition was marginal, indicating that NH3 is the active molecular species. By themselves, neither ambient pH nor ionic strength appeared to affect cohesive performance within the ranges employed. The inhibition was immediately and completely reversed upon removal of NH4Cl or a shift of ambient pH from 7.5 to 6.5. The presence of cycloheximide did not affect the recovery of cohesivity after NH4Cl removal. (2) The presence of 15 mM succinate, propionate, or acetate also reduced cell cohesivity. The timing and extent of the inhibition were identical at pH 7.5 and 6.5. The inhibition was expressed immediately and was reversible. Each of the acids acted synergistically with NH4Cl. The relative potencies of these metabolites acting singly or in combination as inhibitors of cohesivity corresponded roughly to their potencies as modulators of the cAMP relay (Williams et al., 1984). (3) The sensitivity to the metabolites was stage specific, being maximal during and shortly after aggregation and disappearing abruptly at 11-12 hr. This corresponds to the time at which this cohesive system, responsible for the end-to-end cell associations evident during aggregation (H. Beug, G. Gerisch, S. Kempff, V. Riedel, and G. Cremer, 1970, Exp. Cell. Res. 63, 147-158) is supplanted by a newly arisen, serologically and genetically distinct system which thereafter maintains the integrity of the aggregate (C. Steinemann and R.W. Parish, 1980, Nature (London) 286, 721-724; D.K. Wilcox and M. Sussman, 1981, Dev. Biol. 82, 102-112, and Proc. Natl. Acad. Sci. USA 78, 358-362; C.L. Saxe III and M. Sussman, 1982, Cell 29, 755-759). The activities of the metabolites, detailed above, are discussed in relation to their previously demonstrated activities as morphogens.     

Gametogenesis inCoelomomyces dodgei Couch (Blastocladiales,Chytridiomycetes)

Summary The ultrastructure of gametogenesis was studied inCoelomomyces dodgei Couch (Blastocladiales, Chytridiomycetes), an obligate parasite of anopheline mosquito larvae and the copepod,Acanthocyclops vernalis. In infected copepods reared under a 16/8 hours light/dark photoperiod at 25 +2 °C., the gametophyte develops over a period of approximately seven days, and gametogenesis is triggered by the onset of the dark period during the last day of development. The initial step of gametogenesis is the elongation of the centriole to form the kinetosome, and measuring time from the onset of the final dark period (0 hours), this occurs prior to the beginning of the light period (8 hours). Subsequently, small vesicles that appear to originate from elements of the rough endoplasmic reticulum (rER) fuse at the distal end of the kinetosome forming the flagellar vesicle into which the axonemal microtubules elongate to form the flagellum (8–12 hours). Similar small vesicles apparently also derived from rER align in planes and fuse to form cleavage furrows which delineate the gamete initials (12–14 hours). As the gamete initials begin forming, the mitochondria within each initial fuse to form a single mitochondrion that associates with the lipid globules and microbodies forming the microbody-lipid globule complex (12–16 hours). The time elapsed between the formation of the flagellar vesicle to the release of mature gametes from the copepod host is about 8.5 hours. No differences were observed in the processes or timing of gametogenesis in male and female gametophytes.     

Isolation and partial characterization of inner and outer membrane fractions of Nitrobacter hamburgensis

Abstract Highly purified preparations of inner, i.e. cytoplasmic and intracytoplasmic, membranes and outer membranes were isolated from Nitrobacter hamburgensis strain X14 by sucrose density-gradient centrifugation of cell-free extracts. The two membrane fractions differed markedly in morphology, density, and protein composition as determined by polyacrylamide gel electrophoresis. The inner membrane fraction was enriched in NADH oxidase and nitrite oxidase activity. It contained four major protein bands of apparent M _rs of 28 000, 32 000, 70 000, and 116000. The outer membrane fraction was characterized by the presence of 2-keto-3-deoxyoctonate and contained two major proteins of apparent M _rs of 13 000 and 50 000. There was no evidence for differences between cytoplasmic and intracytoplasmic membranes.