Reversed-phase chromatographic method for specific determination of glutathione in cultured malignant cells

A chromatographic method for the specific determination of glutathione in malignant cell lines is described. The method is based on the ability of glutathione-S-transferase to specifically and quantitatively conjugate glutathione to 1-chloro-2,4-dinitrobenzene and chromatographic quantitation of the resultant conjugate, dinitrophenyl-S-glutathione, by reversed-phase liquid chromatography. The assay can be performed on 20 000 g supernatants of cell homogenates without acid extraction. 2-Mercaptoethanol, a sulfhydryl compound often used as a thiol-protective agent to preserve enzymatic activities of a number of enzymes, did not interfere with glutathione determination by this method. The dinitrophenyl-S-glutathione isolated from either standard glutathione samples or from cell homogenates was shown to be identical to authentic dinitrophenyl-S-glutathione using mass spectrometry. Recovery of glutathione in standard samples by the current method was identical to that determined using 5,5′-dithiobis(2-nitrobenzoic acid). Exogenous glutathione added to supernatants of cell homogenate in the presence or absence of 2-mercaptoethanol was also completely recovered.     

Isolation and properties of a natural inhibitor of the chloroplast adenosine triphosphatase

The complete sequence of protein L17 which is a component of the large subunit of the E. coli ribosome has been determined. Peptides deriving from enzymatic hydrolysis with trypsin, thermolysin, chymotrypsin and S. aureus and A. mellea protease were isolated and sequenced by the DABITC/PITC double coupling method. Some overlapping peptides were obtained after mild acid cleavage of the protein. According to the amino acid sequence protein L17 contains 127 residues and has a molecular mass of 14 365. The primary structure of protein L17 agrees well with the amino acid analysis of the intact protein and its N-terminal sequence as derived from automatic sequencing in an improved Beckman sequencer. Secondary predictions and a search for homologous sequence stretches to other ribosomal proteins were made.     

Synthesis of daunosamine-containing disaccharides

Treatment of 2,3,6-trideoxy-1,4-di-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-l-lyxo-hexopyranose (1) with benzyl 2,3-dideoxy-d-glycero-pentopyranoside and p-toluenesulfonic acid gave a mixture of benzyl 2,3,6-trideoxy-4-O-p-nitrobenzoyl-3- (trifluoroacetamido)-l-lyxo-hexopyranoside (49%) and benzyl 2,3-dideoxy-4-O-[2,3,6-trideoxy-4-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-α-l-lyxo-hexopyranosyl]-d-glycero-pentopyranoside (4, 20 %). The structure of the disaccharide 4 was confirmed by a detailed, mass-spectrometric analysis in three modes, namely, negative- and positive-ion, chemical ionization, and electron impact. Similar treatment of the bis(p-nitrobenzoate) 1 with ethyl 2,3-dideoxy-d-glycero-pentopyranoside gave the ethyl glycoside and the desired disaccharide, showing that the transglycosylation is not restricted to benzyl glycosides. Removal of the p-nitrobenzoyl and the benzyl groups from 4 gave the disaccharide 2,3-dideoxy-4-O-(2,3,6-trideoxy-3-trifluoroacetamido-α-l-lyxo-hexopyranosyl)-d-glycero-pentopyranose.     

Synthesis of 7-(tetrahydropyran-2-yl)- and 7-(4-acetoxytetrahydropyran-2-yl)-ε-rhodomycinone and 7-(tetrahydropyran-2-yl)-ε-pyrromycinone

The ¹³C.n.m.r spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of α(1→3)- and α-(1→6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in α-(1→6)-linkages and the latter in α-(1→3)-linkages. The structural conclusions arrived at by ¹³C-n.m.r. spectroscopy are consistent with data from methylation analysis and ¹H-n.m.r. spectroscopy.     

Hemoglobin J Cairo: beta 65 (E9) Lys leads to Gln, A new hemoglobin variant discovered in an Egyptian family.

The present report describes clinical, hematological and biochemical studies of a 27-year old Egyptian woman in whom a fast moving Hb variant was found. The abnormal Hb constituted 48% of the total erythrocyte Hb of the propositus and her father. Structural studies demonstrated that in the abnormal Hb lysine beta 65 is replaced by glutamine. The new Hb mutant is designated hemoglobin J Cairo beta 65 (E9) Lys leads to Gln. This substitution results in only a moderate decrease in cooperativity. No evidence of Hb instability was found. A slight anemic state has been observed in the propositus since she reached adolescence.     

The area of discovery ofApanteles glomeratus (Hymenoptera: Braconidae),Pteromalus puparum (Pteromalidae) andBrachymeria regina (Chalcididae)

The areas of discovery ofApanteles glomeratus, Pteromalus puparum andBrachymeria regina were calculated using two different models. Increasing host or parasite density generally resulted in an initial increase followed by a decrease in area of discovery.