Specific knockdown of m-calpain blocks myogenesis with cDNA deduced from the corresponding RNAi

Fusion of mononuclear myoblast to multinucleated myotubes is crucial for myogenesis. Both µ- and m-calpain are ubiquitously expressed in most cells and are particularly abundant in muscle cells. Knockout of calpain-1 (catalytic subunit of µ-calpain) induced moderate platelet dysaggregation, preserving the normal development and growth, although knockout of calpain-2 (m-calpain) is lethal in mice. Therefore, there should be muscle-specific function of m-calpain per se. Previous methods lack direct evidence for the involvement of m-calpain, because the specific inhibitor to m-calpain has not been developed yet and the inhibition was less potent. Here, we show that screened RNA interference (RNAi) specifically blocked the m-calpain expression by 95% at both the protein and the activity levels. After transfection of adenovirus vector-mediated cDNA corresponding to the RNAi-induced short hairpin RNA, m-calpain in C₂C₁₂ myoblasts was knocked down with no compensatory overexpression of µ-calpain or calpain-3. The specific knockdown strongly inhibited the fusion to multinucleated myotubes. In addition, the knockdown modestly blocked ubiquitous effects, including cell migration, cell spreading, and alignment of central stress fiberlike structures. These results may indicate that m-calpain requiring millimolar Ca²⁺ level for the full activation plays specific roles in myogenesis, independent of µ-calpain, and leave us challenging problems in the future. RNA interference; muscle cell development; fusion; adenovirus vector     

Isolation of uncultivable anaerobic thermophiles of the family Clostridiaceae requiring growth-supporting factors

Novel groups of uncultivable anaerobic thermophiles were isolated from compost by enrichment cultivation in medium with a cell-free extract of Geobacillus toebii. The cell-free extract of G. toebii provided the medium with growth-supporting factors (GSF) needed to cultivate the previously uncultured microorganisms. Twenty-nine GSF-requiring candidates were successfully cultivated, and 16 isolated novel bacterial strains were classified into three different groups of uncultivable bacteria. The similarity among these 16 isolates and a phylogenetic analysis using 16S rRNA gene sequences revealed that these GSF-requiring strains represented novel groups within the family Clostridiaceae.     

Nucleus : cell volume ratio directs the timing of the increase in blastomere adhesiveness in starfish embryos

Blastomeres of starfish embryos begin to increase in adhesiveness after the eighth cleavage and form a monolayered hollow blastula. To investigate factors that affect the timing of the adhesiveness increase, we changed the volume of the cytoplasm or the ploidy of embryos and examined the morphologic changes in the descendent blastomeres during early cleavage stages. In parthenogenetic embryos, in which the ploidy is doubled, the timing of the increase in adhesiveness was accelerated by one cell cycle. In contrast, the timing was delayed by approximately one cell cycle in a large-sized embryo formed by the fusion of an egg and a non-nucleate egg fragment. These two sets of observations are in accord with the expectation from the classical concept that the DNA: cytoplasmic ratio may direct the timing of events in early development. However, observations of small-sized embryos with a reduced amount of cytoplasm were contradictory to the expectation based on the DNA: cytoplasmic ratio; the timing of the increase in adhesiveness in half-sized embryos was almost the same as in control embryos and the timing was delayed by only one cell cycle in quarter-sized embryos. Measurement of the diameters of nuclei showed that the size of nuclei was variable, depending on the stage of development, the volume of cytoplasm and ploidy. We calculated a volume ratio of nucleus to cytoplasm (N: C volume ratio) for tetraploid, large-, half- and quarter-sized embryos. We found that the embryonic cells begin to adhere always when their N: C volume ratio reaches 0.06. A plausible model for the cellular timing mechanism of cell contact is proposed.     

The TPR domain of BepA is required for productive interaction with substrate proteins and the β‐barrel assembly machinery complex

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N‐terminal protease domain and a C‐terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone‐like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the β‐barrel assembly machinery (BAM) driving integration of β‐barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site‐directed in vivo photo‐cross‐linking was used to map the protein–protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane.     

Biochemical properties of a serine protease from Aspergillus flavus and application in dehairing

The proteases are enzymes produced by several filamentous fungi with important biotechnological applications. In this work, a protease from Aspergillus flavus was characterized. The culture filtrate of A. flavus was purified to homogeneity by Sephacryl S-200 column chromatography followed by CM–cellulose. The molecular weight of the purified enzyme was estimated to be approximately 32?kDa by SDS–PAGE. The enzyme hydrolysed BTpNA (N-α-benzoyl-dl-tyrosyl-p-nitroanilide), azo-casein and casein as substrates. Optimal temperature and pH were 55?°C and 6.5, respectively. The enzyme was stimulated by Mg²⁺, Ca²⁺, Zn²⁺ and inhibited by Hg²⁺ and Ag²⁺ and Cu²⁺. The protease showed increased activity with detergents, such as Tween 80 and Triton X, and was stable to the reducing agents, such as β-mercaptoethanol. The protease activity was strongly inhibited in the presence of phenylmethylsulfonyl fluoride, indicating it is a serine protease. The enzyme entrapped in calcium alginate beads retained its activity for longer time and could be reused up to 10 times. The thermostability was increased after the immobilization and the enzyme retained 100% of activity at 45?°C after 60?min of incubation, and 90% of residual activity at 50?°C after 30?min. In contrast, the free enzyme only retained 10% of its residual activity after 60?min at 50?°C. The enzymatic preparation was demonstrated to be efficient in the capability of dehairing without destruction of the hide. The remarkable properties such as temperature, pH and immobilization stability found with this enzyme assure that it could be a potential candidate for industrial applications.     

Indispensable residue for uridine binding in the uridine-cytidine kinase family

Uridine-cytidine kinase (UCK), including human UCK2, are a family of enzymes that generally phosphorylate both uridine and cytidine. However, UCK of Thermus thermophilus HB8 (ttCK) phosphorylates only cytidine. This cytidine-restricted activity is thought to depend on Tyr93, although the precise mechanism remains unresolved. Exhaustive mutagenesis of Tyr93 in ttCK revealed that the uridine phosphorylation activity was restored only by replacement of Tyr93 with His or Gln. Replacement of His117 in human UCK2, corresponding to residue Tyr93 in ttCK, by Tyr resulted in a loss of uridine phosphorylation activity. These findings indicated that uridine phosphorylation activity commonly depends on a single residue in the UCK family.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

The effects of alphaGalCer-induced TCRValpha24 Vbeta11(+) natural killer T cells on NK cell cytotoxicity in umbilical cord blood

Purpose The first objective of this study was to investigate in vitro effects of -galactosylceramide (GalCer) on the proliferation of umbilical cord blood (UCB) natural killer T (NKT) cells and enhancement of their cytotoxicity. The second one is to examine whether purified NKT cells could affect the cytotoxicity of UCB-NK cells either in the presence or absence of dendritic cells (DCs).Methods Mononuclear cells (MNCs) from UCB were cultured for 2 weeks in the presence of IL-2 (100 U/ml), with or without GalCer. The effect of neutralizing monoclonal antibodies (MoAb) against TCRV24 and CD1d was also examined. TCRV24 V11 double positive NKT cells were purified by FACS sorter and then cocultured with syngeneic isolated UCB^–CD56⁺NK cells in either the presence or absence of DCs. The cytotoxicity against various malignant cell targets and cytokine production was determined.Results The addition of GalCer induced human NKT cells to proliferate in UCB-MNCs to a greater extent than in adult PB-MNCs. However, it suppressed the cytotoxic activity against malignant cell targets. Anti-TCRV24 and CD1d MoAb recovered the cytotoxicity by inhibiting the proliferation of UCB-NKT cells. NKT cells cocultured with auto-DCs significantly increased NK cell cytotoxicity against K562, and Raji cells and produced IFN- at much higher levels than UCB-NKT cells alone.Conclusion In UCB samples, GalCer–pulsed DCs and NKT cells acted together to enhance NK cytotoxicity in vitro.