Bacillus sphaericus sensu lato currently consists of seven or more groups of unrelated taxa, one of which is B. sphaericus sensu stricto and another of which is Bacillus fusiformis. Members of two groups (groups 6 and 7), in common with all other B. sphaericus-like organisms, are unable to grow anaerobically or to use common hexoses, pentoses and hexitols as sources of carbon, have G+C contents of 34-36 mol % and form round spores. Groups 6 and 7 can be differentiated from other B. sphaericus-like organisms by low DNA relatedness and by variations in whole-cell fatty acid composition. Unique characteristics of group 6 include the ability to oxidize beta-hydroxybutyrate, the non-requirement for biotin and thiamin and failure to grow in 5% NaCl. Distinctive traits of group 7 include the inability to oxidize pyruvate and a requirement for biotin, thiamin and cystine for growth. These data show that groups 6 and 7 represent two novel species, for which the names Bacillus pycnus sp. nov. and Bacillus neidei sp. nov., respectively, are proposed; the corresponding type strains are NRRL NRS-1691T (= JCM 11075T) and NRRL BD-87T (= JCM 11077T).

3-Hydroxybutyric Acid↗

PubMed2002

kdsA mutations affect FtsZ-ring formation in Escherichia coli K-12.

No one has, as yet, addressed the relationship between the nature of the outer membrane and cell division. kdsA encodes 3-deoxy-D-manno-octulosonic acid (KDO) 8-phosphate synthetase which catalyses the first step in the synthesis of KDO, the linker between lipid A and oligosaccharide of lipopolysaccharide (LPS). Seven temperature-sensitive mutants containing missense mutations in kdsA were affected in the production of KDO and all mutants stopped dividing at 41 degrees C and formed filaments with either one or no FtsZ ring. All observed defects were reversed by the plasmid-borne wild-type kdsA gene. Western blotting analysis, however, demonstrated that the amount of FtsZ protein was not affected by the mutation. The mutants were more susceptible to various hydrophobic materials, such as novobiocin, eosin Y and SDS at 36 degrees C. Methylene blue, however, restored kdsA mutant growth. Plasmid-borne wild-type msbA, encoding a lipid A transporter in the ABC family, partially suppressed kdsA mutation. A mutation of lpxA, functioning at the first stage in lipid A biosynthesis, inhibited both cell division and growth, producing short filaments. These results indicate that the instability of the outer membrane, caused by the defect in KDO biosynthesis, affects FtsZ-ring formation.

Aldehyde-Lyases↗

PubMed2002

A ligand for peroxisome proliferator activated receptor gamma inhibits cell growth and induces apoptosis in human liver cancer cells.

BACKGROUND AND AIMS: Induction of apoptosis of cancer cells through ligands of nuclear hormone receptors (NHRs) is a new approach in cancer therapy. Recently, one of the NHRs, peroxisome proliferator activated receptor gamma (PPARgamma), has been shown to influence cell growth in certain cancer cells although its effect on hepatocellular carcinoma (HCC) has not been analysed. METHODS: Experiments were conducted using three human liver cancer cell lines, PLC/PRF/5, Hep G2 and HuH-7, in vitro. These cells were exposed to troglitazone, a synthetic ligand for PPARgamma, and the effects on cell growth were analysed. RESULTS: Expression of PPARgamma mRNA was detected in all three liver cancer cell lines. Activation of PPARgamma by troglitazone caused a marked growth inhibition in a dose dependent manner in three hepatoma cell lines. The DNA fragmentation ELISA assay and Hoechst 33258 staining revealed that the growth inhibitory effect by adding troglitazone was due to apoptosis of PLC/PRF/5, which strongly expressed PPARgamma. Troglitazone also induced activation of the cell death protease, caspase 3, but not caspase 8, in PLC/PRF/5 cells. However, expression levels of antiapoptotic factor bcl-2 and apoptosis inducing factor bax were not affected. CONCLUSION: Our study showed that PPARgamma was expressed in human liver cancer cells and that the ligand for PPARgamma, troglitazone, inhibited the growth of these cells by inducing apoptosis through caspase 3 activation, indicating that troglitazone could be potentially useful as an apoptosis inducer for the treatment of HCC.

Antineoplastic Agents↗

PubMed2001

Fermi surface sheet-dependent superconductivity in 2H-NbSe2.

High-resolution angle-resolved photoemission spectroscopy was used to study the superconducting energy gap and changes in the spectral function across the superconducting transition in the quasi-two-dimensional superconductor 2H-NbSe2. The momentum dependence of the superconducting gap was determined on different Fermi surface sheets. The results indicate Fermi surface sheet-dependent superconductivity in this low-transition temperature multiband system and provide a description consistent with thermodynamic measurements and the anomalous de Haas-van Alphen oscillations observed in the superconducting phase. The present data suggest the importance of Fermi surface sheet-dependent superconductivity in explaining exotic superconductivity in other multiband systems with complex Fermi surface topology, such as the borides and f-electron superconductors.

Journal Article↗

PubMed2001

Overexpression of hepatocyte growth factor/scatter factor promotes vascularization and granulation tissue formation in vivo.

The effect of hepatocyte growth factor/scatter factor (HGF/SF) during wound healing in the skin was investigated, using HGF/SF-overexpressing transgenic mouse model. Histological analysis of HGF/SF transgenic mouse excisional wound sites revealed increased granulation tissue with marked vascularization. Northern blot analysis demonstrated that, relative to control, vascular endothelial growth factor (VEGF) expression in transgenic skin was significantly higher at baseline and was robustly up-regulated during wound healing. Elevated levels of VEGF protein were detected immunohistochemically, predominantly in endothelial cells and fibroblasts within the granulation tissue of HGF/SF transgenic skin. Serum levels of VEGF were also elevated in HGF/SF transgenic mice. Thus, results from our study suggest that HGF/SF has a significant effect on vascularization and granulation tissue formation during wound healing in vivo, involving with induction of VEGF.

Animals↗

PubMed2001

Functional analysis of the propeptides of subtilisin E and aqualysin I as intramolecular chaperones.

Several proteases require propeptides for the correct folding of their own protease domain. We have recently found that the propeptide from a thermostable subtilisin homolog aqualysin I can refold subtilisin BPN' when added in trans. Here, we constructed chimeric genes with subtilisin E and aqualysin I to attempt the in cis folding of subtilisin E by means of the propeptide of aqualysin I. Our results indicate that the propeptide of aqualysin I can to some extent chaperone the intramolecular folding of the denatured subtilisin E. These results suggest that propeptides in the subtilisin family, despite their sequence diversity, have similar functions. Further, some enzymatic properties of some chimeras in which the subtilisin mature domain is partly swapped with that of aqualysin I were shown to be more similar to those of aqualysin I.

Bacillus subtilis↗

PubMed2001

Folding pathway mediated by an intramolecular chaperone: propeptide release modulates activation precision of pro-subtilisin.

Propeptides of several proteases directly catalyze the protein folding reaction. Uncatalyzed folding traps these proteases into inactive molten-globule-like conformers that switch into active enzymes only when their cognate propeptides are added in trans. Although tight binding and proteolytic susceptibility forces propeptides to function as single turnover catalysts, the significance of their inhibitory function and the mechanism of activation remain unclear. Using pro-subtilisin as a model, we establish that precursor activation is a highly coordinated process that involves synchronized folding, autoprocessing, propeptide release, and protease activation. Our results demonstrate that activation is controlled by release of the first free active protease molecule. This triggers an exponential cascade that selectively targets the inhibitory propeptide in the autoprocessed complex as its substrate. However, a mutant precursor that enhances propeptide release can drastically reduce the folding efficiency by altering the synergy between individual stages. Our results represent the first demonstration that propeptide release, not precursor folding, is the rate-determining step and provides the basis for the proposed model for precise spatial and temporal activation that allows proteases to function as regulators of biological function.

Catalysis↗

PubMed2001

A novel acetyltransferase found in Saccharomyces cerevisiae Sigma1278b that detoxifies a proline analogue, azetidine-2-carboxylic acid.

L-Azetidine-2-carboxylic acid (AZC), a toxic four-membered ring analogue of L-proline, is transported into the cells via proline transporters. It causes misfolding of the proteins into which it is incorporated competitively with L-proline and thereby inhibits the growth of the cells. We recently have discovered, on the chromosome of Saccharomyces cerevisiae Sigma1278b, a novel gene MPR1 required for the resistance of Sigma1278 background strains to toxic AZC. This gene was missing in the particular yeast strain used for the genomic sequence determination. Although the protein sequence was homologous to that of the S. cerevisiae transcriptional regulator, Mpr1p did not affect the expression of genes involved in proline uptake. However, gene expression in Escherichia coli and enzymatic analysis showed that the MPR1 gene encodes a novel AZC acetyltransferase, by which L-proline itself and other L-proline analogues are not acetylated. Mpr1p was considered to be a member of the N-acetyltransferase superfamily based on the results of an Ala-scan mutagenesis through the highly conserved region involved in binding acetyl-CoA in members of the superfamily. Our findings suggest that Mpr1p detoxifies AZC by acetylating it in the cytoplasm. This enzyme might be utilized as a selective marker in a wide variety of organisms, because the cells expressing the MPR1 gene acquire the AZC-resistant phenotype.

Acetylation↗

PubMed2001

"Opening" the ferritin pore for iron release by mutation of conserved amino acids at interhelix and loop sites.

Ferritin concentrates, stores, and detoxifies iron in most organisms. The iron is a solid, ferric oxide mineral (< or =4500 Fe) inside the protein shell. Eight pores are formed by subunit trimers of the 24 subunit protein. A role for the protein in controlling reduction and dissolution of the iron mineral was suggested in preliminary experiments [Takagi et al. (1998) J. Biol. Chem. 273, 18685-18688] with a proline/leucine substitution near the pore. Localized pore disorder in frog L134P crystals coincided with enhanced iron exit, triggered by reduction. In this report, nine additional substitutions of conserved amino acids near L134 were studied for effects on iron release. Alterations of a conserved hydrophobic pair, a conserved ion pair, and a loop at the ferritin pores all increased iron exit (3-30-fold). Protein assembly was unchanged, except for a slight decrease in volume (measured by gel filtration); ferroxidase activity was still in the millisecond range, but a small decrease indicates slight alteration of the channel from the pore to the oxidation site. The sensitivity of reductive iron exit rates to changes in conserved residues near the ferritin pores, associated with localized unfolding, suggests that the structure around the ferritin pores is a target for regulated protein unfolding and iron release.

Amino Acid Motifs↗

PubMed2001

Ion selectivities of the Ca(2+) sensors for exocytosis in rat phaeochromocytoma cells.

1. The ion selectivities of the Ca(2+) sensors for the two components of exocytosis in rat phaeochromocytoma (PC12) cells were examined by measurement of membrane capacitance and amperometry. The cytosolic concentrations of metal ions were increased by photolysis of caged-Ca(2+) compounds and measured with low-affinity indicators benzothiazole coumarin (BTC) or 5-nitrobenzothiazole coumarin (BTC-5N). 2. The Ca(2+)-induced increases in membrane capacitance comprised two phases with time constants of 30--100 ms and 5 s. Amperometric events reflecting the exocytosis of large dense-core vesicles occurred selectively in the slow phase, even with increases in the cytosolic Ca(2+) concentration of > 0.1 mM. 3. The slow component of exocytosis was activated by all metal ions investigated, including Cd(2+) (median effective concentration, 18 pM), Mn(2+) (500 nM), Co(2+) (900 nM), Ca(2+) (8 microM), Sr(2+) (180 microM), Ba(2+) (280 microM) and Mg(2+) (> 5 mM). In contrast, the fast component of exocytosis was activated by Cd(2+) (26 pM), Mn(2+) (620 nM), Ca(2+) (24 microM) and Sr(2+) (320 microM), but was only slightly increased by Ba(2+) (> 2 mM) and Co(2+) and not at all by Mg(2+). 4. The fast component, but not the slow component, was competitively blocked by Na(+) (median effective concentration, 44 mM) but not by Li(+), K(+) or Cs(+). Thus, the Ca(2+) sensor for the fast component of exocytosis is more selective than is that for the slow component; moreover, this selectivity appears to be based on ionic radius, with cations with radii of 0.84 to 1.13 A (1 A = 0.1 nm) being effective. 5. These data support a role for synaptotagmin--phospholipid as the Ca(2+) sensor for the exocytosis of large dense-core vesicles and they suggest that an additional Ca(2+)-sensing mechanism operates in the synchronous exocytosis of synaptic-like vesicles.

Animals↗