Some properties of β-1,3-glucan hydrolyzing enzymes from the rotifer Brachionus plicatilis

We examined some characteristics of hydrolyticenzymes, especially -1,3-glucanase, to obtain theinformation of cell wall lytic enzymes forrotifers.Crude enzyme (ammonium sulfate fraction) of rotifershydrolyzed starch, -1,3-glucan, glycol chitinand CM-cellulose. Optimum pH for hydrolysis ofstarch and CM-cellulose was 6.5, and that for -1,3glucan and glycol chitin was pH 6.0. Pectic acid,xylan and agarose were not hydrolyzed at pH 3–10.-1,3 glucanase was purified about 73-fold from crudeenzyme by ion-exchange chromatography and gelfiltration. Optimum pH and temperature of the enzymewere 6 and 60 °C, respectively. The molecular weight ofthe enzyme was estimated about 260 kDa by gelfiltration. The enzyme was inhibited byHgCl₂ and MnCl_{_2}.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

The static head method for determining the charge stoichiometry of coupled transport systems. Applications to the sodium-coupled D-glucose transporters of the renal proximal tubule

The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented. The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient. The charge stoichiometry can then be calculated from thermodynamic principles. In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time. The charge stoichiometries of two renal sodium coupled D-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined. The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.     

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     

Changes in cell surface charges during differentiation of isolated micromeres and mesomeres from sea urchin embryos

Changes in the negative surface charge were observed by cell electrophoresis during the differentiation of micromeres and mesomeres isolated from 16-cell-stage sea urchin embryos. Micromeres and mesomeres were separated by a sucrose density gradient column and were cultured in normal seawater. An isolated micromere developed to a cell aggregate, and, at the mesenchyme-blastula stage of control, the aggregate began to scatter into single cells. These processes are quite similar to those of the primary mesenchyme cells in situ. An isolated mesomere, on the other hand, developed into an ectodermal vesicle. At desired stages of development, the cell aggregates which derived from single blastomeres were dissociated into single cells, and their electrophoretic mobilities were measured. It was found that the electrophoretic mobility of the micromere- and mesomere-derived cells concomitantly increased from the early blastula stage up to the early mesenchyme stage. In contrast with the mesomere-derived cells, however, the micromere-derived cells showed another increase in electrophoretic mobility when the cells began to migrate as primary mesenchyme cells. These results show that a correlation exists between the increase in cell surface negative charge and the migration of the primary mesenchyme cells.     

Photochemical reaction of 9-cis-retro-γ-rhodopsin at low temperatures

9-cis-Retro-γ;rhodopsin (λ_max = 420 nm) was prepared from 9-cis-retro-γ-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11-cis forms of retro-γ-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-γ-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of bathorhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.     

Ultrastructure of intracytoplasmic membranes of Methanomonas margaritae cells grown under different conditions

The development of intracytoplasmic membranes of Methanomonas margaritae cells grown under different culture conditions was studied. Growth on methane was strongly accelerated by the addition of copper ions. Acceleration by copper, however, was not observed in the case of growth on methanol. Cells grown on methane with copper possessed intracytoplasmic membranes along the cell periphery. When the organism was grown in a medium lacking copper, intracytoplasmic membranes appeared as large vesicles surrounded by a unit membrane at the periphery of the cell. The vesicles originated from paired membranes due to the absence of copper in the medium. Cells grown on methanol with or without copper possessed a number of vesicles of different sizes arranged in a chain along the cell periphery. The possible relationship between membrane arrangement and methane oxidation is discussed.     

New fluorogenic substrates for horseradish peroxidase: Rapid and sensitive assays for hydrogen peroxide and the peroxidase

p-Hydroxyphenyl compounds [3-(p-hydroxyphenyl)propionic acid, p-hydroxyphenethyl alcohol, hordenine, p-ethylphenol, 3-(p-hydroxyphenyl)-1-propanol, p-n-propylphenol, and p-hydroxyphenyllactic acid] were recently found to be excellent fluorogenic substrates for the horseradish peroxidase-mediated reaction with hydrogen peroxide. A very rapid and sensitive method for the fluorometric assays of hydrogen peroxide and the peroxidase was established by using 3-(p-hydroxyphenyl)propionic acid as the best of these substrates; hydrogen peroxide can be assayed precisely in amounts as small as 0.1 nmol, with peroxidase activity as low as 7.8 μU.