Gamma-tubulin distribution during cortical microtubule reorganization at the M/G1 interface in tobacco BY-2 cells

Cortical microtubules are considered to regulate the direction of cellulose microfibril deposition. Despite their significant role in determining cell morphology, cortical microtubules completely disappear from the cell cortex during M phase and become reorganized at G1 phase. The mechanism by which these microtubules become properly formed again is, however, still unclear. We have proposed that the origin of cortical microtubules is on the daughter nuclear surface, but further cortical microtubule reorganization occurs at the cell cortex. Hence it is probable that the locations of microtubule organizing centers (MTOCs) are actively changing. However, the actual MTOC sites of cortical microtubules were not clearly determined. In this paper, we have examined the distribution of gamma-tubulin, one of the key molecules of MTOCs in various organisms, during cortical microtubule reorganization using both immunofluorescence and a GFP reporter system. Using a monoclonal antibody (clone G9) that recognizes highly conserved residues in y-tubulin, y-tubulin was found to be constitutively expressed and to be clearly localized to microtubule structures, such as the preprophase bands, spindles, and phragmoplasts, specific to each cell cycle stage. This distribution pattern was confirmed by the GFP reporter system. During cortical microtubule reorganization at the M to G1 transition phase, gamma-tubulin first accumulated at the daughter nuclear surfaces, and then seemed to spread onto the cell cortex along with microtubules elongating from the daughter nuclei. Based on the results, it was confirmed that daughter nuclear surfaces acted as origins of cortical microtubules, and that further reorganization occurred on the cell cortex.     

Chronic intracerebroventricular infusion of MCH causes obesity in mice. Melanin-concentrating hormone

Melanin-concentrating hormone (MCH) is a cyclic amino acid neuropeptide localized in the lateral hypothalamus. Although MCH is thought to be an important regulator of feeding behavior, the involvement of this peptide in body weight control has been unclear. To examine the role of MCH in the development of obesity, we assessed the effect of chronic intracerebroventricular infusion of MCH in C57BL/6J mice that were fed with regular or moderately high-fat (MHF) diets. Intracerebroventricular infusion of MCH (10 microg/day for 14 days) caused a slight but significant increase in body weight in mice maintained on the regular diet. In the MHF diet-fed mice, MCH more clearly increased the body weight accompanied by a sustained hyperphagia and significant increase in fat and liver weights. Plasma glucose, insulin, and leptin levels were also increased in the MCH-treated mice fed the MHF diet. These results suggest that chronic stimulation of the brain MCH system causes obesity in mice and imply that MCH may have a major role in energy homeostasis.     

The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin,ExoU

Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA(2) and cPLA(2) align or are similar to ExoU sequences. Site-specific mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid profiles and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modification by eukaryotic factors.     

Construction of recombinant hemagglutinin derived from the gingipain-encoding gene of Porphyromonas gingivalis, identification of its target protein on erythrocytes, and inhibition of hemagglutination by an interdomain regional peptide

Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44(720-1081), a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44(720-1138), did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44(720-1138) but not in HGP44(720-1081), could bind HGP44(720-1081) in a dose-dependent manner and effectively inhibited HGP44(720-1081)-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44(720-1081)-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44(720-1081)-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44(720-1081) could bind to asialoglycophorin with a dissociation constant of 3.0 x 10(-7) M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.     

Lysophosphatidic acid stimulates neuronal differentiation of cortical neuroblasts through the LPA1-G(i/o) pathway

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates cortical development. Here we examined how LPA influences the cell fate of cortical neuroblasts using a neurosphere culture system. We generated neurospheres in the presence of basic fibroblast growth factor (bFGF). Treatment with LPA throughout the culture period significantly reduced the number of cells in the neurospheres. When dissociated single cells derived from neurospheres were induced to differentiate by adherence on coverslips, the proportion of MAP2-positive neurons was higher in LPA-treated neurospheres than in those treated with bFGF alone, and the proportion of myelin basic protein-positive oligodendrocytes was lower. Consistent with this finding, LPA raised the ratio of beta-tubulin type III-positive young neurons and reduced the ratio of CD140a-positive oligodendrocyte precursors in neurospheres. These effects of LPA were inhibited by pretreatment of neurospheres with pertussis toxin or an LPA(1)-preferring antagonist, Ki16425. Moreover, LPA-induced enhancement of neuronal differentiation was not observed in neurospheres derived from lpa(1)-null mice. These results suggest that LPA promotes the commitment of neuroblasts to the neural lineage through the LPA(1)-G(i/o) pathway.     

Protocol for rat single muscle fiber isolation and culture

To attain a superior in vitro model of mature muscle fibers, we modified the established protocol for isolating single muscle fibers from rat skeletal muscle. Muscle fiber cultures with high viability were obtained using flexor digitorum brevis muscle and lasted for at least 7 days. We compared the expression levels of adult myosin heavy chain (MyHC) isoforms in these single muscle fibers with myotubes formed from myoblasts; isolated fibers contained markedly more abundant adult MyHC isoforms than myotubes. This muscle fiber model, therefore, will be useful for studying the various functions and cellular processes of mature muscles in vitro.