Sumio Maeda

Stereospecific positioning of the cis-acting sequence with respect to the canonical promoter is required for activation of the ompC gene by a positive regulator, OmpR, in Escherichia coli.

Expression of the ompC gene coding for a major outer-membrane protein of Escherichia coli is regulated by a transcriptional activation mechanism that requires the ompR gene product, OmpR. It was demonstrated that multiple OmpR molecules bind to a cis-acting sequence located upstream from the canonical -35 and -10 regions of the ompC promoter. Using an ompC-lacZ fusion gene, the distance between the cis-acting upstream sequence (OmpR-binding site) and the -35 and -10 regions (RNA polymerase-binding site) has been changed. We demonstrated that the ompC transcription was activated in an OmpR-dependent manner even when the cis-acting upstream sequence was separated from the -35 and -10 regions by several turns of the DNA helix, providing that the distance between them was a near-integral multiple of one turn of the DNA helix. Evidence is presented that stereospecific positioning of the cis-acting upstream sequence with respect to the canonical promoter is required for activation of the ompC gene by the positive regulator, OmpR.

Isolation of rat mitochondrial transcription factor A (r-Tfam) cDNA

We have isolated rat mitochondrial transcription factor A (Tfam; formerly known as mtTFA) cDNA clones from a rat cerebellum cDNA library using human Tfam cDNA as a probe. The deduced amino acid sequence of r-Tfam shows 62% and 89% overall identity to human and mouse Tfam, respectively. We also show the presence of two r-Tfam isoforms in testis as for mouse. Our findings suggest that the mechanisms underlying transcription of mitochondrial genes are conserved among rat, mouse, and human.

Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone.

Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain). In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as ‘cell-to-cell transformation’. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s) that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10–5–10–6, suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria.

INHIBITION OF MITOCHONDRIAL GENE EXPRESSION BY ANTISENSE RNA OF MITOCHONDRIAL TRANSCRIPTION FACTOR A (mtTFA)

Mitochondrial transcription factor A (mtTFA) plays an important role in regulating the expression of mitochondrial genes. To gain a better understanding of the relationships between mitochondrial gene expression and mtTFA, the mtTFA gene was inserted into a mammalian expression vector, both in the sense orientation and in the antisense orientation. After construction, these plasmids were transfected into COS‐7 cells. In antisense‐transformed cells, the expression of the cytochrome c oxidase subunit I and subunit III genes encoded by mitochondrial DNA was inhibited and there was an accompanying reduction of the level of mtTFA protein. These results provide direct evidence that the expression of mitochondrial genes is under the control of mtTFA.

Induction of apoptosis in primary culture of rat hepatocytes by protease inhibitors

There is growing evidence that suggests the involvement of intracellular proteases in the process of apoptosis or programmed cell death. In this study, we have demonstrated that leupeptin, a cysteine protease inhibitor, can significantly increase the incidence of both apoptotic nuclear morphology change and internucleosomal DNA fragmentation in primary cultured hepatocytes in the absence of known apoptotic stimuli for hepatocytes. On the other hand, aspartic and serine protease inhibitors showed little or no effects on the apoptotic changes. In addition, we found that the apoptotic changes could be induced by chloroquine, an inhibitor of lysosomal proteolysis, but could not be induced by calpain inhibitors. These data suggest that inhibition of lysosomal cysteine proteases may induce apoptosis in primary cultured hepatocytes.

Immobilization and characterization of a thermostable β-galactosidase from a thermophilic anaerobe on a porous ceramic support

A thermostable β-galactosidase (EC 3.2.1.23) from a thermophilic anaerobe, strain NA10, was purified from the crude extract of the Escherichia coli transformant harboring the lacN gene. The purified enzyme was physically and covalently immobilized to a porous ceramic support, SM-10. Among the supports tested, the highest residual activity after 3 h incubation at 70° C was obtained when the enzyme was covalently immobilized to silanized SM-10 with 3-[2-(2-amino-ethylaminoethylamino)propyl]trimethoxysilane. The amount of the enzyme immobilized was about 60 mg/g of this support. The enzymatic properties were almost the same as those of the free enzyme. The half-life of this immobilized enzyme was estimated to be approximately 450 h at the pasteurization temperature (65° C).

Influence of aldehyde groups on the thermostability of an immobilized enzyme on an inorganic support

Abstract The influence of glutaraldehyde cross-linking on the thermostability of an immobilized enzyme on an inorganic support is described. A thermostable β-galactosidase derived from a thermophile was covalently linked to a silanized porous inorganic support with glutaraldehyde. The effect of the amount of the immobilized enzyme on its thermostability was investigated at different temperatures and it was found that a combination of a low enzyme/support (g enzyme/g support) and high incubation temperature resulted in a considerable decrease in the residual activity. The thermostability of the immobilized enzyme under these conditions was improved by additional treatment with a solution of basic amino acids after immobilization procedure. These results suggest that the stability at high temperature of an immobilized enzyme by glutaraldehyde cross-linking is remarkably affected by free aldehyde groups remaining on the surface of an inorganic support. Therefore, it is important to eliminate free aldehyde groups for preventing the surface from influencing the enzymes at high incubation temperature.

Identification of a novel DNA element that promotes cell-to-cell transformation in Escherichia coli

Recently, we discovered a novel phenomenon, “cell‐to‐cell transformation” by which non‐conjugative plasmids are transmitted horizontally in co‐cultures of Escherichia coli F− strains. In this study, we aimed to identify the DNA element responsible for the high cell‐to‐cell transformability of pHSG299. By transplanting pHSG299 DNA fragments into pHSG399, a plasmid showing low transformability, we discovered that a specific 88 bp fragment of pHSG299 significantly promoted pHSG399 transformability. Although several short motif‐like repetitive sequences (6–10 bp) were present in the 88 bp sequence, no known DNA motifs were recognized, suggesting that this 88 bp sequence (cell‐to‐cell transformation promoting sequence, CTPS; Accession number: AB634455) is a novel DNA element.

Overproduction of thermostable β-galactosidase in Escherichia coli, its purification and molecular structure

Abstract The lacN gene encoding thermostable β-galactosidase from a thermophilic anaerobe strain NA10 was overexpressed in Escherichia coli strain MV1184 by cloning the gene downstream from the lac promoter on pUC119. The amount of the enzyme produced by the E. coli transformant was estimated to be about 20% of the total cellular proteins when induced with isopropyl-β- d -thiogalactopyranoside. Fed-batch culture of this strain resulted in about a 2,400-fold increase in enzyme production as compared to that in the original bacterium, strain NA10. The enzyme was purified, and its molecular structure was determined by SDS-polyacrylamide gel electrophoresis and gel filtration to be a tetrametric protein consisting of identical subunits.

Mechanisms of active cell death in isolated hepatocytes

Active (or programmed) cell death is a generic term for “suicide-like” cell death. Its critical feature is the dependency on vital cell functions and inherent genetic programming. It occurs in the context of a broad range of physiological functions, including homeostatic regulation of tissue mass, removal of unwanted or harmful cells in immune systems, removal of (pre)neoplastic tissues, and morphogenesis during early development [1–3]. “Apoptosis” is the principal form of active cell death and is generally defined by its characteristic morphological and biochemical changes: nuclear condensation and fragmentation, formation of apoptotic bodies and internucleosomal DNA fragmentation [4,5]. At present, there are growing numbers of reports dealing with the molecular mechanisms of apoptosis (for detailed reviews, see [6–10]). Briefly, stimuli that induce apoptosis vary widely, but their respective signalling pathways frequently converge on one mechanism, in which a few common protein families play important roles: among them are the caspase (previously called the ICE/Ced-3 family) [10,11] and the Bcl-2/Ced-9 [10,12] families. Recent data suggest that mitochondria and cytochrome c play key roles in the regulation of caspases [6,7,9]. In addition, other mechanisms including non-caspase proteases [e.g. 13–18], protein phosphorylation [5,19], redox regulation [20], and Ca2+ signaling [21], may also participate in the process of apoptosis. Other forms of caspase-independent apoptosis [22–25] as well as non-apoptotic forms of active cell death [26–30] have also been reported.