Polymorphism in Nucleotide Sequence of Mitochondrial Intergenic Region in Scleractinian Coral (Galaxea fascicularis)

A region of 826 bp that is unlikely to code for a protein, ribosomal RNA, or transfer RNA was identified between the cytochrome b and NADH–ubiquinone oxidoreductase chain 2 loci in the mitochondrial DNA of the scleractinian reef coral Galaxea fascicularis. Nucleotide sequences were determined in a part (625 bp) of this intergenic region in 95 individuals collected at 9 sites in the Ryukyu Archipelago in southwestern Japan. A total of 8 haplotypes were found, and a deletion of 290 bp was found in 3 of them. Significant differences were found in frequencies of the haplotypes at 3 sampling sites. The presence or absence of the deletion was highly correlated with the hard or soft morphotype. The deletion was found in the majority of hard-type colonies, but in a small fraction of soft-type individuals.     

Coaggregation facilitates interspecies hydrogen transfer between Pelotomaculum thermopropionicum and Methanothermobacter thermautotrophicus

A thermophilic syntrophic bacterium, Pelotomaculum thermopropionicum strain SI, was grown in a monoculture or coculture with a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain DeltaH. Microscopic observation revealed that cells of each organism were dispersed in a monoculture independent of the growth substrate. In a coculture, however, these organisms coaggregated to different degrees depending on the substrate; namely, a large fraction of the cells coaggregated when they were grown on propionate, but relatively few cells coaggregated when they were grown on ethanol or 1-propanol. Field emission-scanning electron microscopy revealed that flagellum-like filaments of SI cells played a role in making contact with DeltaH cells. Microscopic observation of aggregates also showed that extracellular polymeric substance-like structures were present in intercellular spaces. In order to evaluate the importance of coaggregation for syntrophic propionate oxidation, allowable average distances between SI and DeltaH cells for accomplishing efficient interspecies hydrogen transfer were calculated by using Fick's diffusion law. The allowable distance for syntrophic propionate oxidation was estimated to be approximately 2 mum, while the allowable distances for ethanol and propanol oxidation were 16 mum and 32 mum, respectively. Considering that the mean cell-to-cell distance in the randomly dispersed culture was approximately 30 mum (at a concentration in the mid-exponential growth phase of the coculture of 5 x 10(7) cells ml(-1)), it is obvious that close physical contact of these organisms by coaggregation is indispensable for efficient syntrophic propionate oxidation.     

Roles of aromatic residues in the structure and biological activity of the small cytokine,growth-blocking peptide (GBP)

Growth-blocking peptide (GBP) is a small (25 amino acids) insect cytokine with a variety of functions: controlling the larval development of lepidopteran insects, acting as a mitogen for various types of cultured cells, and stimulating insect blood cells. The aromatic residues of GBP (Phe-3, Tyr-11, and Phe-23) are highly conserved in the ENF peptide family found in lepidopteran insects. We investigated the relationship between the biological activities and structural properties of a series of GBP mutants, in which each of the three aromatic residues is replaced by a different residue. The results of the hemocytes-stimulating assays of GBP mutants indicated that Phe-3 is the key residue in this activity: Ala or Tyr replacement resulted in significant loss of the activity, but Leu replacement did not. The replacements of other aromatic residues hardly affected the activity. On the other hand, NMR analysis of the mutants suggested that Tyr-11 is a key residue for maintaining the core structure of GBP. Surprisingly, the Y11A mutant maintained its biological activity, although its native-like secondary structure was disordered. Detailed analyses of the (15)N-labeled Y11A mutant by heteronuclear NMR spectroscopy showed that the native-like beta-sheet structure of Y11A was induced by the addition of 2,2,2-trifluoroethanol. The results suggest that Y11A has a tendency to form a native-like structure, and this property may give the Y11A mutant native-like activity.     

Stimulation of proliferation of rat hepatic stellate cells by galectin-1 and galectin-3 through different intracellular signaling pathways

We found that the expression of galectin-1 and galectin-3 was significantly up-regulated in hepatic stellate cells (HSCs) both in the course of their transdifferentiation into myofibroblasts, a process of "self-activation," and in the fibrosis of liver tissues. Recombinant galectin-1 and galectin-3 stimulated the proliferation of cultured HSCs via the MEK1/2-ERK1/2 signaling pathway. However, galectin-3 utilized protein kinases C and A to induce this process, whereas galectin-1 did not. We also found that thiodigalactoside, a potent inhibitor of beta-galactoside binding, attenuated the effects of both galectins. In addition, galectin-1, but not galectin-3, promoted the migration of HSCs. Thus, it appears that galectin-1 and galectin-3, generated by activated HSCs, could participate in beta-galactoside binding and induce different intracellular signaling pathways leading to the proliferation of HSCs.     

Interleukin-2-collagen chimeric protein which liberates interleukin-2 upon collagenolysis

Interleukin-2 (IL-2) is a potent activator of cellular immunity and has been utilized as an immunotherapeutic agent. We stably immobilized human IL-2 to collagen by covalently binding it to the N-terminus of human type III collagen (3A1) as IL2-3A1 chimeric protein using recombinant technology. The present study was aimed at liberating IL-2 from the immobilized chimeric protein by treating the chimera with bacterial collagenase. These IL2-3A1 chimeras were synthesized in insect cells which had been infected with baculovirus vectors carrying IL2-3A1 cDNA. The IL2-3A1 protein produced was shown to be in a pepsin-resistant triple helical structure and exhibited IL-2 activity to a similar extent as IL-2 itself. IL2-3A1 could be immobilized on the surface of plastic dishes by incubating it in the dishes. The IL-2 region of the immobilized IL2-3A1 was liberated to culture media by collagenase treatment and this freed IL-2 stimulated the growth of lined T cells. Thus, IL2-3A1 chimeric protein could be utilized as an IL-2 deliverer whose T cell mitogenic activity can be liberated by a collagenolytic environment.     

Germ cell lineage from a single blastomere at 8-cell stage in shiro-uo (ice goby)

Shiro-uo (ice goby; teleost fish), Leucopsarion petersii, shows a unique cleavage pattern characterized by two tires of blastomeres at 8-cell stage, like that of echinoderm and amphibian embryo. Such a pattern is suitable to isolation and cell lineage experiments. In this study, cell lineage of germ-line was traced by histological observation and cell labelling experiment at the 8-cell stage. Primordial germ cells (PGCs) were first detected histologically at the 10-somite stage, and migrated to gonadal anlage at 10 days post-fertilization, through usual way described in other teleost species. When a single blastomere was labelled with tracer dye at 8-cell stage, both upper and lower tires generated labelled PGCs at gonadal anlage although upper tires occasionally. This result suggests that all blastomeres at the 8-cell stage have potential to produce PGCs in shiro-uo.     

Potent modification of low density lipoprotein by group X secretory phospholipase A2 is linked to macrophage foam cell formation

The deposition of cholesterol ester within foam cells of the artery wall is fundamental to the pathogenesis of atherosclerosis. Modifications of low density lipoprotein (LDL), such as oxidation, are prerequisite events for the formation of foam cells. We demonstrate here that group X secretory phospholipase A2 (sPLA2-X) may be involved in this process. sPLA2-X was found to induce potent hydrolysis of phosphatidylcholine in LDL leading to the production of large amounts of unsaturated fatty acids and lysophosphatidylcholine (lyso-PC), which contrasted with little, if any, lipolytic modification of LDL by the classic types of group IB and IIA secretory PLA2s. Treatment with sPLA2-X caused an increase in the negative charge of LDL with little modification of apolipoprotein B (apoB) in contrast to the excessive aggregation and fragmentation of apoB in oxidized LDL. The sPLA2-X-modified LDL was efficiently incorporated into macrophages to induce the accumulation of cellular cholesterol ester and the formation of non-membrane-bound lipid droplets in the cytoplasm, whereas the extensive accumulation of multilayered structures was found in the cytoplasm in oxidized LDL-treated macrophages. Immunohistochemical analysis revealed marked expression of sPLA2-X in foam cell lesions in the arterial intima of high fat-fed apolipoprotein E-deficient mice. These findings suggest that modification of LDL by sPLA2-X in the arterial vessels is one of the mechanisms responsible for the generation of atherogenic lipoprotein particles as well as the production of various lipid mediators, including unsaturated fatty acids and lyso-PC.     

Micro-dystrophin cDNA ameliorates dystrophic phenotypes when introduced into mdx mice as a transgene

The adeno-associated virus vector is a good tool for gene transfer into skeletal muscle, but the length of a gene that can be incorporated is limited. To develop a gene therapy for Duchenne muscular dystrophy, we generated a series of rod-truncated micro-dystrophin cDNAs: M3 (one rod repeat, 3.9 kb), AX11 (three rod repeats, 4.4 kb), and CS1 (four rod repeats, 4.9 kb). These micro-dystrophins, driven by a CAG promoter, were used to produce transgenic (Tg) mdx mice and all three micro-dystrophins were shown to localize at the sarcolemma together with the expression of dystrophin-associated proteins. Among them, CS1 greatly improved dystrophic phenotypes of mdx mice and contractile force of the diaphragm in particular was restored to the level of normal C57BL/10 mice. AX11 modestly ameliorated the dystrophic pathology, but, importantly, M3-Tg mdx mice still showed severe dystrophic phenotypes. These data suggest that the rod structure, and its length in particular, is crucial for the function of micro-dystrophin.     

Endoplasmic reticulum calcium release is modulated by actin polymerization

Intracellular calcium ions regulate the structure and functions of cytoskeletal proteins. On the other hand, recent studies have shown that the cytoskeleton, and actin filaments in particular, can modulate calcium influx through plasma membrane ligand- and voltage-gated channels. We now report that calcium release from inositol trisphosphate (IP3) and ryanodine-sensitive endoplasmic reticulum (ER) stores is modulated by polymerization and depolymerization of actin filaments in cultured hippocampal neurons. Depolymerization of actin filaments with cytochalasin D attenuates calcium release induced by carbamylcholine (CCh; a muscarinic agonist for IP3 pathway), caffeine (a ryanodine receptor agonist) and thapsigargin (an inhibitor of the ER calcium- ATPase) in both the presence and absence of extracellular calcium. Conversely, the actin polymerizing agent jasplakinolide potentiates calcium release induced by CCh, caffeine and thapsigargin. Cytochalasin D attenuated, while jasplakinolide augmented, thapsigargin-induced JNK activation and neuronal cell death. Our data show that the actin cytoskeleton regulates ER calcium release, suggesting roles for actin in the various physiological and pathological processes that involve calcium release.     

Identification of a signal transduction switch in the chemokine receptor CXCR1

Chemokine receptors belong to the superfamily of G protein-coupled receptors, which regulate the trafficking and activation of leukocytes, and operate as coreceptors in the entry of HIV-1. To investigate the early steps in the signal transmission from the chemokine-binding site to the G protein-coupling region we engineered metal ion-binding sites at putative extracellular sites in the chemokine receptor CXCR1. We introduced histidines into sites located in the second and third putative extracellular loops of CXCR1, creating single, double, and triple mutant receptors: R199H, R203H, D265H, R199H/R203H, R199H/D265H, R203H/D265H, R203H/H207Q, and R199H/R203H/D265H. Cells expressing the double mutants R199H/D265H and R203H/D265H and the triple mutant R199H/R203H/D265H failed to trigger interleukin 8-dependent calcium responses. Interestingly, calcium responses mediated by the single mutant R203H and the double mutants R199H/R203H and R203H/H207Q were blocked by Zn(II), indicating the creation of a functional metal ion-binding site. On the other hand, cells expressing all single, double, or triple histidine-substituted CXCR1 demonstrated high affinity binding to interleukin 8 in the presence and absence of metal ions. These findings indicate that occupation of the engineered metal-binding site uncouples the chemokine-binding site from the activation mechanism in CXCR1. Most importantly, we identify for the first time elements of an early signal transduction switch of chemokine receptors before the activation of cytoplasmic G proteins.