The order of hepatic cytotoxicity of bile salts in vitro does not agree with that examined in vivo in rats

Using an enzyme release from isolated rat hepatocytes incubated with a bile salt as a marker, the cytotoxic order of bile salts was found to be taurochenodeoxycholate, glycochenodeoxycholate greater than tauroursodeoxycholate, glycoursodeoxycholate, cholate greater than taurocholate, glycocholate. Thus, the cytotoxicity of conjugates of ursodeoxycholate was greater than that of conjugates of cholate. However, these data do not agree with the order of cytotoxicity of these bile salts previously studied in vivo by the authors which demonstrated the least cytotoxic nature of conjugates of ursodeoxycholate.     

Effects of environmental conditions on xylose fermentation by recombinant Escherichia coli

In batch fermentations, optimal conversion of xylose to ethanol by recombinant Escherichia coli was obtained under the following conditions: 30 to 37 degrees C, pH 6.4 to 6.8, 0.1 to 0.2 M potassium phosphate buffer, and xylose concentrations of 8% or less. A yield of 39.2 g of ethanol per liter (4.9% ethanol by volume) was observed with 80 g of xylose per liter, equivalent to 96% of the maximum theoretical yield. Maximal volumetric productivity was 0.7 g of ethanol per liter per h in batch fermentations and 30 g of ethanol per liter per h in concentrated cell suspensions (analogous to cell recycling).     

Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A beta-lactamases.

By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities. Most of the mutants at position 447 retained the binding activity but lost the complementation activity, the exception being the D447E mutant, which retained both. The binding rates for various penicillins of the D447N mutant, which had lost the complementation activity, were almost identical to those of the wild type. The binding of the mutants at position 544 tended to require a higher penicillin concentration, and that of the K544H mutant required a lower pH. When the roles of the counterpart residues, Lys73, Glu166, and Lys234, in class A beta-lactamases were considered, the results suggested that Lys333 and Asp447 are essential for the acylation and acyl-transfer steps, respectively, and that Lys544 stabilizes the Michaelis complex through its side-chain positive charge.     

Protein phylogeny of translation elongation factor EF-1α suggests microsporidians are extremely ancient eukaryotes

Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139     

Endo.SK1: an inducible site-specific endonuclease from yeast mitochondria

Site-specific endonucleases have been found in various eukaryotic organelles such as mitochondria, chloroplasts and nuclei. These endonucleases initiate site-specific or homologous gene conversion in mitochondrial and nuclear DNA. Here, we report a new site-specific endonuclease activity, Endo.SK1, identified in mitochondria of strain SK1, a homothallic diploid strain ofSaccharomyces cerevisiae. Nucleotide sequences around the Endo.SK1-cleavage sites are different from those of known yeast site-specific endonucleases. The Endo.SK1 activity is, at least partly, specified by a gene in the SK1-derived mitochondria. A novel feature of the Endo.SK1 activity is its inducibility: the endonuclease activity was induced by ca. 40-fold by transfer of cells from a glucose medium into an acetate medium, and was then repressed. This transient induction was independent of the ploidy level of the cells, and coincided with induction of fumarase, a mitochondrial enzyme involved in the TCA cycle. Co-induction and co-repression of the mitochondrial site-specific endonuclease activity and a respiration-related enzyme indicate that the endonuclease activity is regulated in response to physiological conditions, and suggest a possible role for the endonuclease in mitochondrial DNA metabolism.     

A point mutation in exon 3 (His 107→Tyr) in two unrelated Japanese patients with carbonic anhydrase II deficiency with central nervous system involvement

We have analyzed two unrelated Japanese patients with carbonic anhydrase II deficiency born to consanguineous parents. We have identified the same mutation as that reported to be homozygous in a Belgian family and compound heterozygous in an American family. It comprises to C-to-T transition that results in the amino acid substitution of Tyr (TAT) for His (CAT) at position 107. This point mutation creates an AccI site that can be conveniently screened by the polymerase chain reaction/restriction fragment length polymorphism method using a restriction enzyme for gene tracking. Our patients exhibit severe mental retardation, not seen in the Belgian and American patients. Received: 23 November 1994 / Revised: 22 May 1995     

Intradermal administration of lipopolysaccharide in treatment of human cancer

 Lipopolysaccharide (LPS) has been recognized as a potent antitumor agent in animal tumor models; however, its use in human cancer therapy has been limited to only one trial, in which LPS from Salmonella was given intravenously. It was not very successful because of poor tumor response and was also toxic. We originally developed LPS prepared from Pantoea agglomerans (LPSp), and this was a well-purified, small-molecular-mass (5 kDa) agent. We chose intradermal rather than intravenous administration in the hope that the former would release LPS slowly into the bloodstream, and thus be less toxic while preserving antitumor activity. In our animal tumor models, intradermal administration was indeed less toxic and more beneficial for tumor regression than intravenous administration. We made a pilot study with intradermal administration of LPSp on the treatment of ten advanced cancer patients. Five of them had evaluable tumor, which had failed earlier to respond to conventional chemotherapy. Cyclophosphamide was also administered in this trial, in anticipation of its synergistic effect with LPSp. In this study LPSp was injected intradermally into each patient twice a week, starting with an initial dose of 0.4 ng/kg, and raising it to 600 or 1800 ng/kg. A 400-mg/m² dose of cyclophosphamide was given intravenously every 2 weeks. After completion of the dose escalation, the treatment was continued for at least 4 months, and it was found that 1800 ng/kg LPSp was well tolerated. A significant level of cytokines was observed in the sera for at least 8 h. These results indicate higher tolerable doses and remarkably more continuous induction of the cytokines than were reported in a previous study by others using intravenous administration. Three of the five evaluable tumors showed a significant response to our combined therapy. Intradermally administered, LPS was less toxic and elicited a tumor response in combination with cyclophosphamide; it can thus can be applied to cancer treatment even in humans. Received: 3 August 1995 / Accepted: 2 April 1996     

A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae

During periods of water deficit, plants accumulate late embryogenesis-abundant (LEA) proteins which are thought to protect cells from stresses associated with dehydration. One of these genes, le25, is expressed in tomato leaves and roots in response to water deficit and abscisic acid accumulation. To study the function of this protein and to test the effect of overproduction of the LE25 protein in Saccharomyces cerevisiae (Sc), a recombinant plasmid in which le25 is expressed under the control of the GAL1 promoter was constructed. The content of LE25 was high in Sc cells transformed with the recombinant plasmid. The transformant exhibited several stress-tolerant phenotypes. Growth of the transformant in a medium with 1.2 M NaCl was improved, as compared to a control strain. While the control strain showed a long lag phase of 40 h, le25-expressing cells showed a shortened lag phase of 10 h. However, no growth improvement was observed in a medium with 2 M sorbitol. In addition, the transformant had an increased survival rate after freezing stress, but not after high-temperature stress. These results, together with its predicted secondary structure, may indicate that LE25 functions as an ion scavenger.     

Studies on the development and three-dimensional reconstruction of giant mitochondria and their nuclei in egg cells ofPelargonium zonale Ait.

Summary The preferential development of giant mitochondria and their nuclei (nucleoids) in the egg cells ofPelargonium zonale Ait. during megasporogenesis and megagametogenesis was examined by fluorescence microscopy, after Technovit embedding and 4,6-diamidino-2-phenylindole (DAPI) staining, fluorimetry for DNA content, using a video-intensified microscope photon-counting system (VIMPICS), and by three-dimensional reconstruction of mitochondrial nuclei (mt-nuclei). Reproductive cells during the megaspore mother cell, meiosis, tetrad, and functioning megaspore stages contained many small mitochondria with characteristic, uniformly DAPI-stained mt-nuclei about 0.3 m in diameter, containing a small amount of DNA (0.3 Mbp). During formation of the 2-, 4-, and 8-nucleate embryo sac, mt-nuclei did not markedly change in shape or DNA content. When the embryo sac formed and differentiation of each cell began, mitochondria and their nuclei in the egg cell took on a small ring or string-like shape. Accompanying the maturation of the embryo sac, they underwent progressive enlargement and gradually altered to long thick strings, or stacks of concentric or half concentric rings. By flower opening, they have developed to an extremely large size. One of these stacks of mt-nuclei was reconstructed in three dimensions; each ring in the stack was cup- or plate-shaped; 5 to 10 rings made up the stack, though each remained discontinuous from the others. From serial sections, we counted 44 mitochondria in one egg cell. Fluorometry using VIMPICS revealed that DNA amount within the stacked mitochondrion increased to 40 times that of the megaspore mother cell stage; a single stack of mitochondria contained 340–1700 Mbp DNA; which means that one egg cell contains at least 15000 Mbp mt-DNA, a value greater than the cell-nuclear DNA content.