Cytoplasmic axial filaments in Escherichia coli cells: possible function in the mechanism of chromosome segregation and cell division.

Overproduction of CafA caused formation of chained cells and minicells. The cafA gene is located downstream from the mre region at 71 min on the Escherichia coli chromosome map and was previously called orfF. A long axial structure running through the chained cells, consisting of bundles of filaments assembled in a long hexagonal pillar several micrometers long and about 0.1 to 0.2 micron in diameter, was visible in both phase-contrast micrographs of the lysozyme-treated cells and electron micrographs of ultrathin sections. The CafA protein displays 34% amino acid similarity with the N terminus of the Ams protein of E. coli, which cross-reacts with antibody to a nonmuscle myosin heavy chain.     

Distribution of serine proteinase inhibitor, clade B, member 6 (Serpinb6) in the adult mouse brain

In the brain, Serpinb6 was identified as an endogenous inhibitor of neuropsin, a member of the S1 (clan SA) family of serine proteases [J. Biol. Chem. 276 (2001) 14562]. In the present study, we investigated the localization of Serpinb6 in the adult mouse brain using in situ hybridization histochemistry and immunohistochemistry. Region-specific patterns of expression were observed and two characteristics were recognized. First, the forebrain limbic area that expressed neuropsin mRNA contained Serpinb6 mRNA at moderate levels but not the lateral septum. On the other hand, Serpinb6 mRNA was also expressed moderately in the substantia nigra-ventral tegmental area system, whose fibers projected to the lateral septum. Additionally, Serpinb6 protein was detected in the lateral septum. Together, it was suggested that the expression of neuropsin in the brain is regulated entirely by Serpinb6. Second, Serpinb6 mRNA and the protein were strongly expressed in most somatic and visceral motoneurons among cranial nerve nuclei. This suggests that another serine protease is regulated by Serpinb6 in motoneurons and/or fibers.     

Expression of HSP72 in the gastric mucosa is regulated by gastric acid in rats-correlation of HSP72 expression with mucosal protection

BACKGROUND AND AIM: The real mechanism of adaptive cytoprotection in the gastric mucosa is not well established. In the present study, we investigated the effect of acid suppressing agents on a 72-kDa heat shock protein (HSP72) expression, which is known as endogenous cytoprotective factor, in the gastric mucosa. Also, the association of gastric mucosal protective function against HCl-challenge was compared between HSP72-induced and -reduced group. MATERIALS AND METHODS: Expression of HSP72 was measured by Western blotting in the gastric mucosa before and after administration of famotidine or omeprazole. The gastric mucosal protective function against 0.6 N HCl was compared between control group and HSP72-reduced group. Also, the effect of increased expression of gastric HSP72 by additional administration of zinc sulfate or zinc L-carnosine, which is known as HSP72-inducer, on mucosal protective function was studied. RESULTS: HSP72 expression in the gastric mucosa was reduced by acid suppressing agents. The lowest expression level of HSP72 was observed 12 h (famotidine, H2-receptor antagonist) or 48 h (omeprazole, proton pump inhibitor) after administration. The gastric mucosal protective ability against 0.6 N HCl was also reduced when HSP72 expression was decreased by famotidine or omeprazole. This phenomenon was reversed by HSP72 induction by additional administration of zinc derivatives. CONCLUSION: Our results might indicate that the expression of HSP72 in the gastric mucosa is physiologically regulated by gastric acid, and that HSP72 induction could be important in view of mucosal protection especially when HSP72 expression is reduced by administration of acid suppressing agents such as proton pump inhibitor or H2 receptor antagonist.     

Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+

Only graminaceous monocots possess the Strategy II iron (Fe)-uptake system in which Fe is absorbed by roots as an Fe3+-phytosiderophore. In spite of being a Strategy II plant, however, rice (Oryza sativa) contains the previously identified Fe2+ transporter OsIRT1. In this study, we isolated the OsIRT2 gene from rice, which is highly homologous to OsIRT1. Real-time PCR analysis revealed that OsIRT1 and OsIRT2 are expressed predominantly in roots, and these transporters are induced by low-Fe conditions. When expressed in yeast (Saccharomyces cerevisiae) cells, OsIRT2 cDNA reversed the growth defects of a yeast Fe-uptake mutant. This was similar to the effect of OsIRT1 cDNA. OsIRT1- and OsIRT2-green fluorescent protein fusion proteins localized to the plasma membrane when transiently expressed in onion (Allium cepa L.) epidermal cells. OsIRT1 promoter-GUS analysis revealed that OsIRT1 is expressed in the epidermis and exodermis of the elongating zone and in the inner layer of the cortex of the mature zone of Fe-deficient roots. OsIRT1 expression was also detected in the ccompanion cells. Analysis using the positron-emitting tracer imaging system showed that rice plants are able to take up both an Fe3+-phytosiderophore and Fe2+. This result indicates that, in addition to absorbing an Fe3+-phytosiderophore, rice possesses a novel Fe-uptake system that directly absorbs the Fe2+, a strategy that is advantageous for growth in submerged conditions.     

Fragmentation of Penicillin-binding Protein-1Bs of Escherichia coli by Proteolytic Enzymes: A Preliminary Study on the Location of the Penicillin-binding Site on the Bifunctional Peptidoglycan Synthetase Protein

The absolute configurations of fenvalerate and other related cyanohydrin esters were studied by circular dichroism (CD) measurements and by high performance liquid chromatography (HPLC). Fenvalerate has UV absorption peaks around 278 nm associated with the ¹L_b phenyl transitions and corresponding positive CD peaks were observed around 281 nm for the enantiomers of (S)-configuration at the cyanohydrin chiral center. Most of the other cyanohydrin esters also gave positive CD peaks for the enantiomers of (S)-configuration. CD spectra in the 180 to 250 nm range were also studied.

By HPLC, the elution order of the diastereoisomers of cyanohydrin esters were closely correlated with their absolute configuration and the (RS,SR)-pair consistently eluted earlier than the (RR,SS)-pair for α-substituted phenylacetic acid esters.     

Supersensitivity to β-Lactam Antibiotics in Escherichia coli Caused by D-Alanine Carboxypeptidase IA Mutation

Escherichia coli cells acquired supersensitivity to various β-lactam antibiotics by dacA mutation, a defect in D-alanine carboxypeptidase IA activity. The mutant cells were rather less sensitive to mecillinam than the dacA⁺ cells. This mutation did not result in either thermosensitivity of cell growth or appreciable increase of the generation times in usual rich media, but the resulting appearance of supersensitivity to β-lactam antibiotics suggests that the cell wall or envelope of this mutant is somewhat abnormal and thus that D-alanine carboxypeptidase IA is involved in cell wall or envelope synthesis.     

The role of PKC isoforms in the inhibition of NF-κB activation by vitamin K2 in human hepatocellular carcinoma cells

Vitamin K (VK) has diverse protective effects against osteoporosis, atherosclerosis and carcinogenesis. We recently reported that menatetrenone, a VK2 analogue, suppressed nuclear factor (NF)-κB activation in human hepatoma cells. Although NF-κB is regulated by isoforms of protein kinase C (PKC), the involvement of PKCs in VK2-mediated NF-κB inhibition remains unknown. Therefore, the effects of VK2 on the activation and the kinase activity of each PKC isoform were investigated. The human hepatoma Huh7 cells were treated with PKC isoform-specific inhibitors and/or siRNAs against each PKC isoform with or without 12-O-tetradecanoylphorbol-13-acetate (TPA). VK2 inhibited the TPA-induced NF-κB activation in Huh7 cells. NF-κB activity was inhibited by the pan-PKC inhibitor Ro-31-8425, but not by the PKCα-specific inhibitor Gö6976. The knockdown of individual PKC isoforms including PKCα, δ and ? showed only marginal effects on the NF-κB activity. However, the knockdown of both PKCδ and PKC?, together with treatment with a PKCα-specific inhibitor, depressed the NF-κB activity. VK2 suppressed the PKCα kinase activity and the phosphorylation of PKC? after TPA treatment, but neither the activation nor the enzyme activity of PKCδ was affected. The knockdown of PKC? abolished the TPA-induced phosphorylation of PKD1, and the effects of PKD1 knockdown on NF-κB activation were similar to those of PKC? knockdown. Collectively, all of the PKCs, including α, δ and ?, and PKD1 are involved in the TPA-mediated activation of NF-κB. VK2 inhibited the NF-κB activation through the inhibition of PKCα and ? kinase activities, as well as subsequent inhibition of PKD1 activation.     

Identification of an FBN1 mutation in bovine Marfan syndrome-like disease

Mutations in the gene encoding fibrillin-1 (FBN1), a component of the extracellular microfibril, cause Marfan syndrome (MFS). Frequent observation of cattle with a normal withers height, but lower body weight than age-matched normal cattle, was recently reported among cattle sired by phenotypically normal Bull A, in Japanese Black cattle. These cattle also showed other characteristic features similar to the clinical phenotype of human MFS, such as a long phalanx proximalis, oval face and crystalline lens cloudiness. We first screened a paternal half-sib family comprising 36 affected and 10 normal offspring of Bull A using the BovineSNP50 BeadChip (illumina). Twenty-two microsatellite markers mapped to a significant region on BTA10 were subsequently genotyped on the family. The bovine Marfan syndrome-like disease (MFSL) was mapped onto BTA10. As FBN1 is located in the significant region, FBN1 was sequenced in Bull A, and three affected and one normal cattle. A G>A mutation at the intron64 splicing accepter site (c.8227-1G>A) was detected in 31 of 36 affected animals (84.7%). The c.8227-1G>A polymorphism was not found in 20 normal offspring of Bull A or in 93 normal cattle unrelated to Bull A. The mutation caused a 1-base shift of the intron64 splicing accepter site to the 3' direction, and a 1-base deletion in processed mRNA. This 1-base deletion creates a premature termination codon, and a 125-amino acid shorter Fibrillin-1 protein is produced from the mutant mRNA. We therefore conclude that the c.8227-1G>A mutation is causative for MFSL. Furthermore, it was suggested that Bull A exhibited germline mosaicism for the mutation, and that the frequency of the mutant sperm was 14.9%.     

Slr2019, lipid A transporter homolog,is essential for acidic tolerance in Synechocystis sp. PCC6803

Photosynthesis Research - Living organisms must defend themselves against various environmental stresses. Extracellular polysaccharide-producing cells exhibit enhanced tolerance toward adverse...     

Strong enhancement of recombinant cytosine deaminase activity in Bifidobacterium longum for tumor-targeting enzyme/prodrug therapy

In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity.In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.