Enhancement of Nitrogen Dioxide-Induced Lipid Peroxidation and Dna Strand Breaking by Cysteine and Glutathione

Nitrogen dioxide less than 100 ppm in air induced lipid peroxidation of liposome composed of l-palmitoyl-2-arachidonylphosphatidylcholine as assessed by thiobarbituric acid reactivity. The nitrogen dioxide-induced lipid peroxidation was enhanced by cysteine, glutathione and bovine serum albumin. While the activity of nitrogen dioxide in air to induce single strand breaks of supercoiled plasmid DNA was low, the breaking was remarkably enhanced by cysteine, glutathione and bovine serum albumin. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that certain strong oxidant(s) were generated by interaction of nitrogen dioxide and cysteine. The spin trapping using 3,5-dibromo-4-nitrosobenzene-sulfonate suggested that sulfur-containing radicals were generated by interaction of nitrogen dioxide and cysteine or glutathione. Hence, certain sulfur-containing radicals generated by the interaction which could effectively induce lipid peroxidation and DNA strand breaks.     

Biosynthesis of various types of collagen by human hepatoma cells in vitro

A cloned human hepatoma cell line (HH2-1) produced and formed collagen fibers in vitro. The relative rate of collagen synthesis by the cells was increased with an enhancement of the cell density. An analysis of the components of the collagen using sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the cells synthesized interstitial collagen, types I and III, and other collagenous proteins. Thus, human hepatoma cells may play an important role in the formation of stromal collagen in the tumor.     

Digestibility of the hydrogenated derivative of an isomaltooligosaccharide mixture by rats.

The digestibility of the hydrogenated derivative of an isomaltooligosaccharide mixture (IMO-H) was investigated. In an in vitro experiment, the digestibility of IMO-H was examined by models of the digestive system. IMO-H was resistant to two types of alpha-amylase and to artificial gastric juice. Enzymes in the rat small intestinal mucosa hydrolyzed tri-, tetra- and higher saccharide alcohols to disaccharide alcohol, removing successive glucose units from the non-reducing ends of the chains. The hydrolysis ratio for IMO-H was intermediate between the values for maltose and maltitol. In an in vivo study, growing rats were fed on an experimental diet containing IMO-H, maltitol, or hydrogenated palatinose in the range from 5% to 20%. The growth parameters of the rats fed on the test sugar show that the availability of IMO-H was about 1.2 to 1.25 times that of maltitol or hydrogenated palatinose.     

Beta-sheet folding of fragment (16-36) of bovine pancreatic trypsin inhibitor as predicted by Monte Carlo simulated annealing.

A tertiary structure prediction is described using Monte Carlo simulated annealing for the peptide fragment corresponding to residues 16-36 of bovine pancreatic trypsin inhibitor (BPTI). The simulation starts with randomly chosen initial conformations and is performed without imposing experimental constraints using energy functions given for generic interatomic interactions. Out of 20 simulation trials, seven conformations show a sheet-like structure--two strands connected by a turn--although this sheet-like structure is not as rigid as that observed in native BPTI. It is also shown that these conformations are mostly looped and exhibit a native-like right-handed twist. Unlike the case with the C-peptide of RNase A, no conspicuous alpha-helical structure is found in any of the final conformations obtained in the simulation. However, the lowest-energy conformation does not resemble exactly the native structure. This indicates that the rigid beta-sheet conformation of native BPTI merely corresponds to a local minimum of the energy function if the fragment with residues 16-36 is isolated from the native protein. A statistical analysis of all 20 final conformations suggests that the tendency for the peptide segments to form extended beta-strands is strong for those with residues 18-24, and moderate for those with residues 30-35. The segment of residues 25-29 does not tend to form any definite structure. In native BPTI, the former segments are involved in the beta-sheet and the latter in the turn. A folding scenario is also speculated from this analysis.     

Characterization of transverse tubule membrane proteins: tentative identification of the Mg-ATPase

Vesiculated fragments of chicken skeletal muscle transverse tubule (TT) membranes were analyzed for their content of loosely associated and integral membrane proteins. Of particular interest was the identification of the magnesium-stimulated ATPase (Mg-ATPase), which is characteristically located in native isolated TT vesicles of chicken skeletal muscle [R. A. Sabbadini and V. R. Okamoto (1983) Arch. Biochem. Biophys. 223, 107-119]. A number of the proteins found in vesicular TT preparations were found to be extractable by a mild Triton-X100 treatment and were identified as aldolase, enolase, creatine kinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and pyruvate kinase. Approximately 60% of TT-associated protein was extracted with Triton, resulting in a twofold enrichment of the Mg-ATPase. Concommitantly, one core integral membrane protein possessing a Mr of 102,000 was enriched, suggesting that it is responsible for the Mg-ATPase activity present in chicken skeletal muscle TT membranes.     

Fractionation of chromosomal proteins of pea seedlings using procedures which avoid denaturation

Chromatin was prepared from the buds and cotyledons of Alaskapea seedlings. The dissociated chromosomal components in thepresence of 2 M NaCl and 5 M urea were completely fractionatedinto DNA and proteins with a Bio-Gel A50 column. The proteinswere recovered by (NH₄)₂SO₄ and further fractionated into histonesand non-histone proteins using a Bio-Rex 70(Na⁺) column. Thedifference in the ratios of histones to non-histone proteinsbefore and after chromatography with the Bio-Rex 70 was lessthan 10%. The histones and non-histone proteins thus preparedshowed typical protein absorption spectra. Polyacrylamide gelelectrophoresis of histones showed that the histone compositionsin buds and cotyledon were similar, but the amount of HI histoneswas a little less in cotyledons than in buds. Unlike histones,non-histone proteins fractionated by SDS-polyacrylamide gelelectrophoresis indicated distinct differences between the twotissues. Buds had more heterogeneous non-histone proteins, atleast 13 polypeptides, than cotyledons did. On the other hand,non-histone proteins of cotyledons showed less heterogeneityand lacked proteins of high molecular weight which were foundin buds. (Received May 6, 1976; )     

Acetazolamide inhibits the recovery from triethyl tin intoxication: Putative role of carbonic anhydrase in dehydration of central myelin

The vacuolar degeneration of central myelin was produced in Sprague-Dawley rats by oral administration of triethyl tin. The wet weight of brain stems which seems to reflect the degree of accumulation of water increased during the administration of the toxin, whereas the activity of 2, 3-cyclic nucleotide 3-phosphodiesterase altered less remarkably. When TET was withdrawn from the drinking water, the rats showed a dramatic clinical improvement along with reduction in wet weight of brain stems. Treatment with acetazolamide following TET inhibited the clinical improvement and reduction in wet weight of brain stems. The present results indicates that central myelin has plasticity in recovering from the vacuolar degeneration by removing the accumulated fluid and carbonic anhydrase is possibly involved in the dehydration, of myelin in such a recovery phase.     

A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity

A plasmid encoding a mutant gene of heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, was induced by treatment of plasmid EWD 299 with hydroxylamine. A mutant strain of E. coli HB 101 carrying the mutant plasmid pTUH 6A produced a low toxic LT analogue (mutant LT), which was cross-reactive with anti-LT antibody. The mutant LT activity was less than 0.15 and 0.006% of the normal LT in the rabbit ileal loop test and in the rabbit skin permeability test, respectively. The amino acid composition of the mutant LT-B subunit was the same as that of the normal B subunit. Though the A2 fragment of the mutant LT was identical to normal LT by DNA analysis, the A1 fragment of the mutant LT differed from the normal A1 fragment in one amino acid at position 112; namely it had lysine instead of glutamic acid from the N terminus. These data suggest that glutamic acid at position 112 from the N terminus of the A1 fragment is important for the A subunit to express its biological activity.     

Molecular cloning and nucleotide sequence of a gene for alkaline cellulase from Bacillus sp. KSM-635

A gene for alkaline cellulase from the alkalophilic Bacillus sp. KSM-635 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. Although the recombinant plasmid contained two HindIII inserts of 2.6 kb and 4.0 kb, the inserts were found to be contiguous in the Bacillus genome by hybridization analysis. Nucleotide sequences of a 2.4 kb region which was indispensable for the production of cellulase, and the flanking, 1.1 kb region, were determined. There was an open reading frame (ORF) of 2823 bp in the 3498 bp sequence determined, which encoded 941 amino acid residues. Two putative ribosome-binding sites and a sigma 43-type, promoter-like sequence were found upstream from an initiation codon in the ORF. The deduced amino-terminal sequence resembles the signal peptide of extracellular proteins. A region of amino acids, 249 to 568, of the deduced amino acid sequence of the cellulase from this organism is homologous with those of alkaline and neutral enzymes of other micro-organisms, but nine amino acid residues were found to be conserved only in the alkaline enzymes.