The amino acid sequences of the cytochromes c553 from Porphyridium cruentum and Aphanizomenon flos-aquae

The amino acid sequences of cytochrome c₅₅₃ from the eukaryotic red alga Porphyridium cruentum and from the prokaryotic cyanobacterium Aphanizomenon flos-aquae have been determined from the tryptic and cyanogen bromide peptides. The results indicate that a charged region of these proteins has evolved with special rapidity to accomodate a rapid evolution of a binding site in the P₇₀₀ electron acceptor complex.     

Cross reactivity and enzyme sensitivity of immunoaffinity purified Sm/RNP antigens

Immunoaffinity purified Sm/RNP antigens from buffalo and goat liver were studied to determine the role of RNA and proteins towards the antigenicity of Sm and RNP antigens. A more direct approach using enzyme-linked immunosorbent assay on nylon beads has been utilized to look into the problem. The effect of enzyme treatment and the role of RNA and protein fractions in influencing antigenicity have been described. RNA seems to be involved in the maintenance of RNP specific polypeptides in suitable conformation so as to keep them in solution. Removal of RNA leads to insolubilization of RNP specific polypeptides. Antibodies to Sm and RNP antigens have been shown to cross react with poly A containing heterogeneous nuclear ribonucleoprotein with no cross reactivity with thymus RNA or DNA.     

Minimum inhibitory concentration of drugs againstMycobacterium leprae as determined by anin vitro assay

The observations that liveMycobacterium leprae after entry into cultured peritoneal macrophages from mice, reduced the EA rosetting macrophages, have been exploited to determine the minimum inhibitory concentration of diamino diphenyl sulphone and rifampicin. Diamino diphenyl sulphone showed a minimum inhibitory concentration of 0.028 μg/ml and rifampicin 0.11μg/ml when given externally. However, there was accumulation of diamino diphenyl sulphone inside the macrophages. At an external concentration of 0.028 μg/ml the concentration inside the macrophage was 0.5μg/ml. The minimum inhibitory concentration for diamino diphenyl sulphone in this assay system is higher by several folds and that for rifampicin is slightly lower, than what is reported earlier with mice foot pad experiments. The minimum inhibitory concentration reported in this assay system is quite close to what is observed forin vitro inhibition ofMycobacterium lufu with both the drugs     

Gonadotropin releasing hormone in first trimester human placenta: Isolation,partial characterisation andin vitro biosynthesis

Using a specific radioimmunoassay for gonadotropin releasing hormone, the presence of gonadotropin releasing hormone like material in the first trimester human placenta has been demonstrated. The material has been partially characterized using carboxy methyl cellulose chromatography, high pressure gel permeation chromatography and reverse phase C18 high pressure liquid chromatographic analysis. Analysis for bioactivity revealed that placental gonadotropin releasing hormone is much more active than synthetic gonadotropin releasing hormone inin vitro rat pituitary lutinising hormone release assay.In vitro biosynthetic studies using labelled precursors and immunoaffinity chromatography indicated that first trimester human placenta synthesizes gonadotropin releasing hormone like material.     

Antigenic determinants distinctive of Methanospirillum hungatei and Methanogenium cariaci identified by monoclonal antibodies

Monoclonal antibodies were prepared against two species of Methanomicrobiaceae. Antibody 1A is specific for Methanospirillum hungatei strain JF1 and the determinant it recognizes is expressed on the surface of JF1 cells, where it is exposed and accessible to antibody. The determinant is found in a polypeptide (MW<12,000) in the sheath that covers the bacterial cell; it is not present in Methanospirillum hungatei strain GP1; and it is not expressed on the surface of whole cells of the other 24 methanogenic bacteria tested. It is therefore a marker of strain JF1, consequently, antibody 1A is potentially useful for tracking JF1 and fragments thereof in a variety of samples. Antibody 7A is specific for Methanogenium cariaci JR1c. It did not react with any other methanogen tested, not even with Mg. marisnigri or Ms. hungatei JF1, although these cross-react with Mg. cariaci if tested with polyclonal antisera. Therefore antibody 7A recognizes specifically a marker of Mg. cariaci JR1c.Abbreviations SIA slide immunoenzymatic assay - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

Comparison of the Ames assay and the induction of sister chromatid exchanges: Results with ten pharmaceuticals and five selected agents

Seven antischistosomal drugs, two antimalarial drugs, and one antiamoebic drug were tested in all five Ames strains for induction of mutation, as well as for induction of cytotoxicity, inhibition of cellular progression, and the induction of sister chromatid exchanges in two cultured mammalian cell lines. We found that two agents shown to be negative in the Ames test were positive for sister chromatid exchange induction. Based on qualitative and quantitative evaluation, we find that all but three of the pharmaceuticals should be considered to be potential human carcinogens.Abbreviations AA 2-aminoanthracene - 9AACC 9-aminoacridine - AM amoscanate - BrdUU bromodeoxyuridine - CA chloroquine diphosphate - CHO Chinese hamster ovary - CQ chloroquine - DAPI 46-diamidino-2-phenylindole - DHY dehydroemetine - DMSO dimethyl sulfoxide - EB ethidium bromide - FCS fetal calf serum - FN 4-fluoro-3-nitrophenyl azide - HY hycanthone - ICP inhibiting cell progression - LU lucanthone - MEM minimal essential medium - 2NF 2-nitrofurantoin - 4NPD 4-nitro-O-phenylenediamine - NZ niridazole - OL oltipraz - OX oxaminiquine - PBS phosphate buffered saline - PQ primaquine - PZ praziquantel - SA sodium azide - SCE sister chromatid exchange     

Photolabeling of staphylococcal alpha-toxin from within rabbit erythrocyte membranes

Intrinsic membrane proteins of rabbit red blood cells were labeled with the photoreactive amphipatic reagent 12-(4-azido-2-nitrophenoxy) stearoyl (1-14C) glucosamine, which inserts into the hydrophobic membrane region and generates a reactive nitrene upon ultraviolet irradiation. Photolabeling of membrane-bound staphylococcal alpha-toxin after lysis of probe-treated rabbit red blood cells by this toxin implies its penetration into the hydrophobic region of the outer leaflet of the membrane. In contrast clostridial theta-toxin and staphylococcal delta-toxin were not labeled, but extraction of intrinsic membrane proteins by delta-toxin was evidenced.     

Aerobic ferrisiderophore reductase assay and activity stain for native polyacrylamide gels

The reduction of ferric iron from microbial iron-binding compounds (siderophores) releases the iron from the siderophore so that it may be utilized by the microorganism. A method to detect aerobic ferrisiderophore reductase activity using ferrozine as a ferrous iron trap is shown to be applicable to cytoplasmic fractions from Rhodopseudomonas sphaeroides and four other different species of bacteria. The ferrisiderophore reductase uses reduced nicotinamide cofactors as reducing agents, and activity is stimulated by flavins. This assay has been adapted as a staining method to locate ferrisiderophore reductase activity in native polyacrylamide gels.     

The use of amphotericin B to detect inhibitors of cellular cholesterol biosynthesis

Pores formed in the membranes of animal cells by complexes of sterols and the polyene antibiotic amphotericin B can efficiently kill the cells. Thus, in the absence of exogenous sources of cholesterol, inhibitors of enzymes in the cholesterol biosynthetic pathway render cells resistant to amphotericin B. Preincubation of Chinese hamster ovary cells with compactin or 25-hydroxycholesterol, inhibitors of the synthesis of the key intermediate mevalonate, protected cells from amphotericin B killing and this protection was reversed by the addition of exogenous mevalonate. The ability of compactin to confer amphotericin B resistance on normal cells was abolished when cells were provided exogenous cholesterol by the receptor-mediated endocytosis of low density lipoprotein. Low density lipoprotein receptor-defective Chinese hamster ovary cells were not subject to this low density lipoprotein-dependent amphotericin B killing. Exogenous mevalonate did not prevent 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, an inhibitor of mevalonate conversion to sterols, from protecting cells from amphotericin B. A simple two-step protocol in which cells are preincubated (15-24 h) with potential inhibitors and then treated (3-6 h) with amphotericin B was devised to provide a sensitive method for detecting direct (e.g., competitive) and regulatory inhibitors of cholesterol biosynthesis. This protocol may prove useful in detecting potential antihypercholesterolemia drugs and is currently being used to isolate mutants in receptor-mediated endocytosis.