13C and 1H NMR studies of helix-coil transition of poly(β-benzyl-L-aspartate) and poly(γ-benzyl-L-glutamate): Behavior in nonprotonating solvent mixtures,and origin of solvent-induced chemical shift

From the results of ¹³C-nmr measurement of poly(β-benzyl-L -aspartate) and its model compounds in dimethyl sulphoxide/deuterated chloroform mixtures, it was found that the side chain of poly(β-benzyl-L -aspartate) is solvated by dimethyl sulphoxide in the region more than dimethyl sulphoxide 20% (v/v), where the backbone maintains the α-helix. The chemical shift differences in the benzyl group carbons of poly(γ-benzyl-L -glutamate) (trifluoroacetic acid/deuterated chloroform) accompanied by the helix-coil transition, originate from the interaction between the ester group of the side chain and trifluoroacetic acid. The chemical shift difference in the ester carbon is similar. On the other hand, the chemical shift differences of the side-chain carbons in the alkyl portion (C^β, C^γ) originate not only from the interaction between the ester group of the side chain and trifluoroacetic acid, but also from some other unknown factors. The chemical shift differences of the side-chain carbons of poly(β-benzyl-L -aspartate) originate from the interaction between the ester group of the side chain and trifluoroacetic acid.     

Production of itaconic acid by Aspergillus terreus immobilized in polyacrylamide gels

Summary Living Aspergillus terreus cells were entrapped in polyacrylamide gels and employed in both replacement batch and continuous column reactors to produce itaconic acid from glucose.With the replacement batch reactor, maximum itaconic acid productivity was observed under the following conditions: pH 2.50, temperature at 35°C, addition of NH₄H₂PO₄ and MgSO₄·7H₂O. Using the continuous reactor, the maximum itaconic acid yield was 60 mg/h/40 g of gel. The biocatalyst activity or half-life was about 10 days.     

Methylamine facilitates demonstration of specific uptake of diphtheria toxin by CHO cell and toxin-resistant CHO cell mutants

We have measured the specific uptake of ¹²⁵I-labelled diphtheria toxin in the presence of methylamine by a number of cell lines with different sensitivities to diphtheria toxin. The results show a strong correlation between the toxin sensitivities of the cell lines and the amount of specific uptake. The specific association of labelled toxin with cells was clearly demonstrated even with CHO cells, a cell line with relatively low sensitivity. Thus, CHO cell mutants that are resistant to diphtheria toxin could be classified as toxin-binding or non-binding cells by this method.     

Complete primary structure of a galactose-specific lectin from the venom of the rattlesnake Crotalus atrox. Homologies with Ca2(+)-dependent-type lectins

The complete primary structure of a galactose-specific lectin contained in the venom of the rattlesnake, Crotalus atrox, was determined. The lectin is composed of two covalently linked, identical subunits, each consisting of 135 amino acid residues. Under physiological conditions the lectin proved to be highly aggregated. The venom lectin contained 9 half-cystines, 8 of which formed four intrasubunit disulfide bridges (Cys3-Cys14, Cys31-Cys131, Cys38-Cys133, and Cys106-Cys123), while Cys86 was involved in an intersubunit disulfide bridge. Because of the high content of disulfide bridges, the intact lectin was extremely resistant to tryptic digestion. The determined amino acid sequence was found to be homologous with those of the so-called carbohydrate recognition domains of Ca2(+)-dependent-type lectins in animal. Among them, 8 amino acid residues (Cys31, Gly69, Trp92, Pro97, Cys106, Asp120, Cys123, and Cys131) were completely conserved. Leu40, Trp67, and Trp81 were also well conserved. The rattlesnake venom lectin showed high hemagglutinating activity. These results, together with the occurrence of similar lectins in crotalid venoms, suggest that these lectins have evolved in order to make the venom a more effective weapon to capture prey animals.     

The micronucleus test using peripheral blood reticulocytes from methotrexate-treated mice.

The induction of micronuclei by methotrexate (MTX) was examined in two laboratories using mouse peripheral blood reticulocytes. MTX was a weak inducer in the micronucleus test using bone marrow cells and single treatments, and was one of the few chemicals showing a multiple-treatment effect (CSGMT/JEMS.MMS, 1990). In our preliminary experiments, the ratio of reticulocytes to total erythrocytes decreased greatly after a single treatment with MTX at 100 mg/kg, so lower dose levels of MTX were selected to carry out the micronucleus test in peripheral blood. Full-scale tests were performed at dose levels of 0, 10, 20, 40, and 80 mg/kg, with five sampling times of 0, 24, 48, 72, and 96 h. Frequencies of micronucleated reticulocytes (MNRETs) increased dose-dependently at 72 h, to a maximum of approximately 1%; some preparations obtained from the animals at higher doses could not be examined because the ratio of reticulocytes to total erythrocytes had decreased severely. At doses of 0.5-4.0 mg/kg, the effect of multiple treatments vs. single treatments was not clear, nor was the maximum level of response much different. Since MTX induced a clear positive response in peripheral blood reticulocytes after a single treatment, the reticulocytes in peripheral blood seem a more sensitive target.     

Biosynthesis of poly(3-hydroxyalkanoate) from amino acids

It was found that an optically active copolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), denoted as P(3HB-co-3HV), is synthesized by Alcaligenes eutrophus H16 from several amino acids under various fermentation conditions. The optimum condition for the biosynthesis from one amino acid, threonine, was investigated and its biosynthetic pathway was discussed on the basis of the relation between the fermentation condition and the co-monomer composition of the produced polyesters.     

Murine acute leukemia cell line with megakaryocytic differentiation (MK-8057) induced by whole-body irradiation in C3H/He mice: cytological properties and kinetics of its leukemic stem cells

Five cases of murine leukemia with megakaryocytic differentiation were observed among the 417 cases of radiation-induced leukemias which developed in 30% of C3H/HeMs mice exposed at 8 to 10 weeks to 0.5 to 5 gy total body irradiation. Cells from individual leukemic colonies in the spleen of the irradiated mice, and cells from colonies in methylcellulose (MC) culture in vitro, derived from one of these leukemias, MK-8057, were injected into mice; both types of cells caused the deaths of the recipient mice by inducing the same type of leukemia. MK-8057 can be maintained in Dexter-type liquid culture with a feeder layer of irradiated bone marrow cells. There was a linear reciprocal relationship between the increasing number of MK-8057 cells injected versus the survival of the recipient mice. A reciprocal relationship also was seen between an increasing number of leukemic stem cells, corresponding to the number of MK-8057 cells, and the survival of mice injected with MK-8057. Giant nuclear megakaryocytes developed during the course of colony growth in the spleen as they did in the MC culture. Such megakaryocytes were acetylcholinesterase positive, whereas leukemic cells in the peripheral blood showed no sign of platelet production nor of a positive reaction to acetylcholinesterase. Cells maintained in culture were entirely positive in platelet glycoprotein IIb/IIIa when anti-human antibody was used. The larger cells in a splenic cell suspension derived from a moribund mouse were separated and enriched by velocity sedimentation using centrifugal elutriation (CE), and then subjected to flow cytometry using propidium iodide staining. Cells with up to 32N-DNA content were detected. After separating MK-8057 by counter-flow CE, the larger cell fraction (mode at 540 microns3) produced more leukemic colonies when injected into irradiated mice than did the small cell fraction (mode at 240 microns3). A higher percent of the larger cell fraction (61.9%) was killed by the addition of tritiated thymidine cytocide than in the smaller cell fraction (14.9%). Thus, the smaller cell fraction is considered to have more leukemic spleen colony-forming units (L-CFU-s) in the resting state.     

Tumor necrosis factor antagonizes inhibitory effect of azidothymidine on human immunodeficiency virus (HIV) replication in vitro.

Tumor necrosis factor alpha (TNF-alpha) completely reverses the activity of azidothymidine (AZT) against human immunodeficiency virus type 1 (HIV-1) in MOLT-4 cell cultures. The 50% effective concentration of AZT, required to protect MOLT-4 cells against the cytopathic effect of HIV-1, increased from 5.8 nM in the absence of TNF-alpha to greater than 125 microM in the presence of TNF-alpha (100 U/ml). TNF-alpha also antagonized the anti-HIV-1 activity of dideoxycytidine but did not markedly affect the anti-HIV-1 activity of dextran sulfate. The intracellular phosphorylation pattern of AZT was not changed upon the presence of TNF-alpha.