Cloning and Sequencing of Two New Verotoxin 2 Variant Genes of Escherichia coli Isolated from Cases of Human and Bovine Diarrhea

We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT-IIv (VT2vp1) and SLT-IIva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin-producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada.     

Effects of detergents on the secondary structures of prion protein peptides as studied by CD spectroscopy.

Pathogenic prion proteins (PrP(Sc)) are thought to be produced by alpha-helical to beta-sheet conformational changes in the normal cellular prion proteins (PrP(C)) located solely in the caveolar compartments. In order to inquire into the possible conformational changes due to the influences of hydrophobic environments within caveolae, the secondary structures of prion protein peptides were studied in various kinds of detergents by CD spectra. The peptides studied were PrP(129-154) and PrP(192-213); the former is supposed to assume beta-sheets and the latter alpha-helices, in PrP(Sc). The secondary structure analyses for the CD spectra revealed that in buffer solutions, both PrP(129-154) and PrP(192-213) mainly adopted random-coils (approximately 60%), followed by beta-sheets (30%-40%). PrP(129-154) showed no changes in the secondary structures even in various kinds of detergents such as octyl-beta-D-glucopyranoside (OG), octy-beta-D-maltopyranoside (OM). sodium dodecyl sulfate (SDS), Zwittergent 3-14 (ZW) and dodecylphosphocholine (DPC). In contrast, PrP(192-213) changed its secondary structure depending on the concentration of the detergents. SDS, ZW, OG and OM increased the alpha-helical content, and decreased the beta-sheet and random-coil contents. DPC also increased the alpha-helical content, but to a lesser extent than did SDS, ZW, OG or OM. These results indicate that PrP(129-154) has a propensity to adopt predominantly beta-sheets. On the other hand, PrP(192-213) has a rather fickle propensity and varies its secondary structure depending on the environmental conditions. It is considered that the hydrophobic environments provided by these detergents may mimic those provided by gangliosides in caveolae, the head groups of which consist of oligosaccharide chains containing sialic acids. It is concluded that PrP(C) could be converted into a nascent PrP(Sc) having a transient PrP(Sc) like structureunder the hydrophobic environments produced by gangliosides.     

In vivo cross-linking of nucleosomal histones catalyzed by nuclear transglutaminase in starfish sperm and its induction by egg jelly triggering the acrosome reaction.

A histone heterodimer, designated as p28, which contains an Nepsilon(gamma-glutamyl)lysine cross-link between Gln9 of histone H2B and Lys5 or Lys12 of histone H4, is present in starfish (Asterina pectinifera) sperm. Treatment of sperm nuclei with micrococcal nuclease produced soluble chromatin, which was size-fractionated by sucrose-gradient centrifugation to give p28-containing oligonucleosome and p28-free mononucleosome fractions, indicating that the cross-link is internucleosomal. When sperm nuclei were incubated with monodansylcadaverine, a fluorescent amine, in the presence or absence of Ca(2+), histone H2B was modified only in the presence of Ca(2+). Gln9, in the N-terminal region, was modified, but the other Gln residues located in the internal region were not, suggesting that the modification takes place on the surface of the nucleosome core by the in situ action of a Ca(2+)-dependent nuclear transglutaminase. Treatment of sperm with the egg jelly, which activates Ca(2+) influx to induce the acrosome reaction, resulted in a significant elevation of the p28 content in the nucleus. This is the first demonstration of an in vivo activation of transglutaminase leading to the formation of a cross-link in intracellular proteins.     

A nuclear gene, erd1, encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up-regulated during senescence in Arabidopsis thaliana

A cDNA, ERD1, isolated from one-hour-dehydrated plants of Arabidopsis thaliana L. encodes a putative protein that is similar to the regulatory ATPase subunit (ClpA) of the Clp protease and contains a putative chloroplast-targeting transit-peptide at the N-terminus. A chimeric gene with the putative plastid-targeting sequence of the erd1 gene fused to the synthetic green-fluorescent protein (sGFP) gene was constructed and introduced into Arabidopsis protoplasts. The N-terminal region of the ERD1 protein directed the sGFP protein into the plastids of the protoplasts, and functioned as a transit peptide. Northern blot analysis indicated that expression of the erd1 gene was induced not only by water stress, such as dehydration and high salinity, but also by natural senescence and dark-induced etiolation. The erd1 gene was not strongly induced by exogenous abscisic acid. A chimeric gene with the 0.9 kb promoter region of the erd1 gene fused to the β-glucuronidase (GUS) reporter gene was constructed, and tobacco plants transformed with the construct. The GUS reporter gene driven by the erd1 promoter was induced by dehydration and high salt stress at significant levels in the transgenic plants. The GUS gene was strongly expressed in older leaves without dehydration, and was induced by dark-induced etiolation. Furthermore, GUS activity was reduced by cytokinin treatment during dark-induced etiolation. These results indicate that expression of the erd1 gene is developmentally up-regulated by senescence as well as by water stress.     

Mammalian expression of single chain variable region fragments dimerized by Fc regions

To promote application of a single chain variable region fragment (sFv) in immunoglobulins, a sFv gene was connected to an IgG1 Fc gene, designated as a sFvc gene, and used for transfection of Sp2/0. As a result, the sFvc protein was found to be secreted in a dimeric form. It is thus felt that the sFvc protein, which mimicks the shape of a naturally occurring antibody, can be simple and useful to reproduce divalency and Fc-associated effecter functions as seen in a natural antibody.Abbreviations Abbreviations sFv single chain variable region fragment - Fc constant region of immunoglobulin - sFvc single chain variable region fragment with an Fc region     

Levamisole in postoperative adjuvant immunochemotherapy for gastric cancer

Summary The usefulness of LMS in postoperative immunochemotherapy of gastric cancer was investigated. In compliance with the protocol, MMC was given at a dose of 20 mg on the day of gastrectomy, and an additional 10 mg on the next day IV. The patients receiving 600 mg Tegafur daily were then divided into two groups according to whether LMS was also given or not. LMS was administered for 3 days before the operation in a daily dose of 150 mg and for 1 year or more after operation according to a schedule of 3 days' administration followed by an 11-day interval. The 2-year follow-up demonstrated that in stage III patients, the LMS (+) regimen was superior to the LMS (–) regimen, since the former prolonged the relapse-free interval significantly. The survival rate for stage III disease was also significantly higher in the LMS (+) than in the LMS (–) group. There was no significant difference in the incidence of subjective or objective side-effects between two groups. The incidence of agranulocytosis was comparable in the two groups.Gastrointestinal Cancer Research Group, Japan Levamisole Research AssociationChairmen of the Gastrointestinal Cancer Research Group, Japan LMS Research AssociationController of the Gastrointestinal Cancer Research Group, Japan LMS Research AssociationMembers of the Data Collection and Analysis SubcommitteeThis study was carried out by the Gastrointestinal Cancer Research Group, Japan LMS Research Association (directed by Prof. Kiyoshi Inokuchi, Dept. of Surgery, Kyushu University and Prof. Eiro Tsubura, Dept. of Internal Medicine, Tokushima University). The results were presented in part at the 19th General Meeting of the Japanese Society for Gastroenterological Surgery in February, 1982     

Photosynthetic regeneration of ATP using a strain of thermophilic blue-green algae

Photosynthetic ATP accumulation was shown in the presence of exogenous ADP plus orthophosphate on illumination to the intact cells of a strain of thermophilic blue-green algae isolated from Matsue hot springs, Mastigocladus sp. Kinetic studies of various effectors on the ATP accumulation proved that the ATP synthesis depends mainly on the cyclic photophosphorylation system around photosystem I (PS-I) in the algal cells. The temperature and pH optima for the accumulation were found at 45 degrees C and pH 7.5. Maximum yield was obtained with light intensity higher than 15 mW/cm(2). Borate ion exerted pronounced enhancement on the ATP synthesis. With a continuous reactor at a flow rate of 1 ml/hr at 45 degrees C and pH 7.5, efficient photoconversion of ADP (2mM, at substrate reservoir) to ATP (1mM, at product outlet) has been maintained for a period of 2.5 days, though the efficiency has decreased in a further 2-day period to the level of 0.5mM ATP/9.5 h of residence time.     

Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein,and enhancement of its accumulation by abscisic acid in somatic embryos

ECP31, an embryogenic-cell protein from carrot (Daucus carota L.), was purified by sequential column-chromatographic steps and digested by V8 protease on a nitrocellulose membrane. The resultant peptides were separated by reverse-phased column chromatography and sequenced. The sequences obtained were 70–80% homologous to those of a late-embryogenesis-abundant protein (D34) from cotton (Baker et al, 1988, Plant Mol. Biol. 11, 227–291). The level of ECP31 in somatic embryos of carrot was increased by treatment of the embryos with 3.7 · 10^–6 M abscisic acid (ABA) for 48 h, and there was no change in this enhanced level for up to 192 h in the presence of ABA. No similar enhancing effect of ABA was observed on the level of ECP31 in embryogenic callus or segments of carrot hypocotyls. In an immunohistochemical analysis, ECP31 was found in epidermal tissue and in the vascular system of ABA-treated somatic embryos.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LEA protein late-embryogenesis-abundant protein To whom correspondence should be addressedThis work was supported in part by a grant-in-aid for Special Research in Priority Areas (Project No. 02242102) from the Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government.