An approach to the identification of adenosine''s inhibitory site on adenylate cyclase

The separate and combined effects of molybdate and dithiothreitol on the stability of human uterine 9 S estrogen receptor were studied. Maximal, short-term, protection of the 9 S estrogen receptor was achieved by the joint inclusion of both stabilizing agents in cytosol buffers. This molybdate—dithiothreitol-mediated stability was dependent on reducing agent concentration inferring sulphydryl involvement in 9 S receptor protection by molybdate. The study also showed that molybdate—dithiothreitol could not prevent the gradual decay of the 9 S estrogen receptor to the 4 S form in cytosols stored at 4°C over prolonged periods.     

An obligatory role of protein glycosylation in the life cycle of yeast cells

Two water-soluble carbodiimides, differing in molecular dimensions, have been used to characterize the cytochrome c binding site of bovine heart cytochrome c oxidase. Several polypeptide components of the enzyme contain acidic residues which are modified by these reagents. Carboxyl groups present in subunit II, VII and polypeptide c, are protected from modification when cytochrome c, equimolar to oxidase, is added and they can cross-link to the substrate once activated by the carbodiimide. Comparison of the modification patterns suggest that the most reactive residues are located on subunit II and VII, the former being also more exposed. The data obtained indicate that even though subunit II plays the major role in binding cytochrome c, at least two other lower Mr polypeptides contribute to the cytochrome c binding domain.     

Factor-free and one-factor-promoted poly(U,C)-dependent synthesis of polypeptides in cell-free systems from Escherichia coli

α-Tropomyosin from rat cardiac muscle was shown by two-dimensional gel electrophoresis to become phosphorylated when tissue slices were incubated in Eagle's medium supplemented with ³²P_i. In the adult rat and mouse heart the level of phosphorylation was ～30%, but the level was much higher in the foetal heart (60–70%). A similar developmental trend was observed in skeletal muscle from the rat and mouse, where phosphorylated forms of both α- and β-tropomyosins were observed. When rat cardiac cells were grown in tissue culture in the presence of ³²P_i, radioactivity was incorporated into the region of the gel containing tropomyosin.     

Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10⁶ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 μM) is then added directly to the growing culture and AS-resistant (AS^r) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT^? clones. The AS^r phenotype is characterized both physiologically and biochemically. All AS^r clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.     

The mutagenic potency of 1,8-dinitropyrene in cultured mouse lymphoma cells

Although non-toxic, 1,8-dinitropyrene (1,8-DNP) was mutagenic for mouse lymphoma L5178Y cells when assayed for induced resistance to 6-thioguanine, methotrexate, ouabain and 1-β-D-arabinofuranosyl cytosine. In bacteria, nitropyrenes are potent inducers of frame-shift mutations, and the induction of ouabain-resistant mutants, believed to be due to base-pair substitutions, suggests that the mechanism of action may be different in mouse cells and bacteria. Long treatment times were required to detect 1,8-DNP-induced mutants in L5178Y cells, suggesting the possibility of an inducible activation system. 4-Nitroquinoline 1-oxide was both toxic and mutagenic to these same 4 mutation assays after short (2 h) treatment times. The dilemma that exists when comparing the mutagenic potential of test chemicals when concentration of mutagen, treatment times and toxicity are markedly different, is discussed.     

Myelin Gangliosides in Vertebrates

Abstract: A phylogenetic survey of brain myelin ganglioside patterns and concentrations has been carried out on 16 vertebrate species. Gangliosides were isolated from purified myelin and found to vary in concentration from 25 μg of sialic acid per 100 mg of myeh for goldfish to a value of 395 for turkey. The latter species had approximately equivalent amounts of G_M1 and G_M4 as the two major gangliosides. The 11 mammals studied all had G_M1 as the major ganglioside, with variable amounts of G_M4; rhesus monkey and human had 20-25% G_M4, whereas the others had less than 10%. Amphibia and fish myelin contained the least total ganglioside, with patterns that showed relatively little G_M1 and no detectable G_M4. Alligator myelin was unique in having a total concentration as high as the avian species, but a pattern with predominantly diand trisialo gangliosides.     

Steady-state kinetic studies on the actin activation of skeletal muscle heavy meromyosin subfragments: Effects of skeletal,smooth and non-muscle tropomyosins

The addition of either smooth muscle or brain tropomyosin to skeletal muscle actoheavy meromyosin (HMM) or acto-myosin subfragment-1 (SF1) produces an activation of the actin-activated ATPase activity up to 100%. This contrasts with the opposite, inhibitory effect produced by skeletal muscle tropomyosin. The degree of activation or inhibition depends on the ionic conditions, which influence the affinities of tropomyosin and HMM or SF1 for actin as well as on the molar ratio of actin to myosin.Enzyme kinetic analysis indicates that the inhibitory effect of skeletal muscle tropomyosin results from an approximately six- to tenfold increase in the apparent affinity (K_app) of the myosin head for the F-actin-tropomyosin complex with a concomitant six- to tenfold reduction in the maximal turnover rate (V_max). Thus, there is no direct competition of skeletal muscle tropomyosin and myosin for the same site on actin. Brain tropomyosin has an opposite effect, decreasing the apparent affinity with concomitant increase in the V_max.The effect of smooth muscle tropomyosin is more complex. At high ratios of myosin to actin this tropomyosin produces the same change in the K_app as skeletal muscle tropomyosin but yields a value of V_max that is about twofold higher. At lower molar ratios (below about 1 to 5 myosin subfragments to actin) the activating effect of this tropomyosin remains unchanged while the apparent affinity decreases to that observed for pure F-actin.On the basis of these data as well as from experiments carried out at fixed actin and varying SF1 concentrations, it is concluded that tropomyosins act in general as allosteric un-competitive inhibitors or activators of actomyosin by increasing or reducing the co-operative activation of myosin by actin at the level of product release.     

Composition and Metabolism of Gangliosides in Rat Peripheral Nervous System During Development

Abstract: Ganglioside composition of rat trigeminal nerve was studied during development in order to understand the changes that occur as a result of cellular differentiation in the nerve. The ganglioside composition of the trigeminal nerve was entirely different from that of brain. The major gangliosides in adult trigeminal nerve were GM₃, GD₃, and LM₁ (sialosyl-lactoneotetraosylceramide or sialosylparagloboside). The structure of LM₁ and other gangliosides was established by enzymatic degradation and by analysis of the products of acid hydrolysis. At 2 days after birth, when the Schwann cells were immature, GM₃ and GD₃ were the major gangliosides in the nerve, 50 and 18 mol %, respectively. As the nerve developed and Schwann cells proliferated and myelinated the axons, the mol % of GM₃ and GD₃ reduced and that of LM₁ steadily increased. Polysialogangliosides did not change drastically with nerve development. The rate of deposition of LM₁ in the nerve with age was very similar to that of myelin marker lipids, cerebrosides, and sulfatides; thus, deposition appears to be localized mainly in the rat nerve myelin. LM₁ also had long-chain fatty acids 22:0 and 24:0, which are not usually found in CNS gangliosides. The ganglioside pattern of the rat trigeminal nerve was very similar to that of rat sciatic nerve, but was different from that of rabbit and chicken sciatic nerve. The activity of the two key enzymes involved in the metabolism of GM₃, viz., CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase and UDP-N-acetylgalactosamine:GM₃-N-acetylgalactosaminyltransferase, was also studied during development of the nerve and brain. The developmental profiles of both enzymes were consistent with the amounts of GM₃ present in the nerve.     

Evolutionary basis and ecological role of toxic microbial secondary metabolites

This presentation develops a theory of the evolutionary origin and ecological implications of toxic microbial secondary metabolites. The theory is based on a model system that outlines cause—effect associations between pertinent biotypes in the aflatoxin contamination of developing maize kernels. The model suggests that the aflatoxin-producing fungi are natural digestive tract inhabitants of a number of insect species that feed on developing kernels. During feeding, the insect larvae introduce fungal propagules and provide infection sites on damaged kernels. The fungal association with insects exhibits extraordinary variability, ranging from symbiotic to pathogenic. Elaboration of aflatoxin by the fungus facilitates the pathogenic process in host insects. The theory contends that genetic information for secondary microbial metabolites evolved during ecosystem disequilibria. During periods of ecological stability, mechanisms evolved for repression of toxic secondary metabolite biosynthesis. The theory broadly suggests that contemporary agricultural activities presents the requisite milieu for production or toxic microbial secondary metabolites.     

A comparison of aflatoxin B1-induced cytotoxicity,mutagenicity and prophage induction in Salmonella typhimurium mutagen tester strains TA1535, TA1538, TA98 and TA100

Treatment of Ames mutagen tester strains with aflatoxin B1 (AFB1) and S9 mix results not only in the production of a poten mutagen, but induces a pathway that leads to the induction of prophages present in all Ames tester strains.Characterization of the prophage induction and mutagenic response following AFB1 treatment showed that plasmid pKM101 dramatically enhances mutagenesis, but suppressed prophage induction. Spontaneous release of phage by TA98 and TA100 was also lower than in TA1535 and TA1538.In addition to mutagenesis and prophage induction, survival of all 4 tester strains was quantitated after AFB1 treatment. The data show that the frameshift tester strains (TA1538 and TA98) are more sensitive to the bactericidal action of AFB1 than the base-pair tester strains (TA1535 and TA100), survival being significantly affected above 100 ng. One of several hypotheses examined was the difference in the number and types of prophages present in base-pair tester strains that are not detectable in the frame-shift tester strains.These data suggest that prophage induction can detect DNA damage that is non-mutagenic; and that it is important to characterize the lysogenic nature of the Ames strains since it may influence the observed histidine revertant rate and the survival of the tester strain.