The study of X-rays and TCDD effects on satellite associations may suggest a simple model for application in environmental mutagenesis

Summary Satellite associations were used as parameters to test nucleolar organizer activity. Assuming that toxic and/or mutagenic agents may affect the ribosomal genes, satellite associations in human lymphocytes were analysed following exposure to X-rays and compared with the satellite association pattern of subjects exposed to TCDD. A significant decrease in the satellite association frequency in D group chromosomes was found both in irradiated lymphocytes and in subjects exposed to Dioxin. The findings seem to be in accordance with the hypothesis based on random damage of functional nucleolar organizing regions.     

Decreased virulence in stable,acapsular mutants ofCryptococcus neoformans

Six acapsular strains ofCryptococcus neoformans obtained by chemical mutagenesis failed to produce a capsulein vivo and were avirulent in mice following high dose intramuscular, intraperitoneal or intravenous inoculation. Peritoneal granulomas were observed in all animals inoculated with the acapsular mutants. These granulomas were characterized by a large central mass consisting of intact, degenerating and necrotic yeast cells. This was surrounded by concentric layers of a broad band of histiocytes, a narrow band of fibroblasts, and around the periphery, a mass of lymphocytes and plasma cells. These isolates did not revert to an encapsulated or virulent state after more than a year of subculturing or 18 passages through mice.     

High performance liquid chromatography,chemical laboratory practice: By Heinz Engelhardt. Springer-Verlag,New York/Berlin, 1979. 248 pp. $29.80

A gel filtration method has been developed for the complete removal of sodium dodecyl sulfate (SDS) from proteins and peptides. The protein or peptide (20 μg–10 mg) containing SDS (up to 30–60 mg) is dissolved in a mixture of propionic acid, formic acid, and water (2:1:2, $\text{v}\text{v}$). Under these conditions, protein-SDS (or peptide-SDS) complexes, as well as SDS micelles, are dissociated. Subsequently, protein and SDS can be separated on a small Sephadex G-25 superfine column. The recovery of protein is typically 90% or more.     

Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem.

In Escherichia coli, two enzymes catalyze the synthesis of methionine from homocysteine using methyltetrahydrofolate as the donor of the required methyl group: cobalamin-dependent and cobalamin-independent methionine synthases. Comparison of the mechanisms of these two enzymes offers the opportunity to examine two different solutions to the same chemical problem. We initiated the research described here to determine whether the two enzymes were evolutionarily related by comparing the deduced amino acid sequences of the two proteins. We have determined the nucleotide sequence for the metE gene, encoding the cobalamin-independent methionine synthase. Our results reveal an absence of similarity between the deduced amino acid sequences of the cobalamin-dependent and cobalamin-independent proteins and suggest that the two have arisen by convergent evolution. We have developed a rapid one-step purification of the recombinant cobalamin-independent methionine synthase (MetE) that yields homogeneous protein in high yield for mechanistic and structural studies. In the course of these studies, we identified a highly reactive thiol in MetE that is alkylated by chloromethyl ketones and by iodoacetamide. We demonstrated that alkylation of this residue, shown to be cysteine 726, results in complete loss of activity. While we are unable to deduce the role of cysteine 726 in catalysis at this time, the identification of this reactive residue suggests the possibility that this thiol functions as an intermediate methyl acceptor in catalysis, analogous to the role of cobalamin in the reaction catalyzed by the cobalamin-dependent enzyme.     

Acetate production from lactate and glucose fermentation by Gluconobacter oxydans

Summary The production of acetate from the fermentation of lactate by Gluconobacter oxydans was studied. Batch experiments showed that glucose was the preferred substrate compared to lactate. A fed-batch culture was fed with a mixture of glucose and lactate followed by periodic addition of lactate. The maximum productivity of acetate was 0.16 g/l h but this value decreased during the fedbatch culture due to growth inhibition by acetate.     

Reactivity and binding of benzo(a)pyrene diol epoxide to poly(dG-dC).(dG-dC) and poly(dG-m5dC).(dG-m5dC) in the B and Z forms

The physical and covalent binding of the carcinogen benzo(a)pyrene-7,8-diol-9,10-oxide (BaPDE) to poly(dG-dC).(dG-dC) and poly(dG-m5dC).(dG-m5dC) in the B and Z forms were studied utilizing absorbance, fluorescence and linear dichroism techniques. In the case of poly(dG-dC).(dG-dC) the decrease in the covalent binding of BaPDE with increasing NaCl concentration (0.1-4 M) as the B form is transformed to the Z form is attributed to the effects of high ionic strengths on the reactivity and physical binding of BaPDE to the polynucleotides; these effects tend to obscure differences in reactivities with the B and Z forms of the nucleic acids. In the case of poly(dG-m5dC).(dG-m5dC) the B-to-Z transition is induced at low ionic strength (2 mM NaCl + 10 microM Co(NH3)6Cl3) and the covalent binding is found to be 2-3-times lower to the Z form than to the B form. Physical binding of BaPDE by intercalation, which precedes the covalent binding reaction, is significantly lower in the Z form than in the B form, thus accounting, in part, for the lower covalent binding. The linear dichroism characteristics of BaPDE covalently bound to the Z and B forms of poly(dG-m5dC).(dG-m5dC) are consistent with nonintercalative, probably external conformations of the aromatic pyrenyl residues.     

Characteristics of different cytoplasmic and nuclear estrogen receptors appearing with continuous hormonal exposure

Biochemical properties of cytosol estrogen receptor (ERC) and nuclear estrogen receptor (ERN) from rat uteri continuously exposed in vivo to 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2) for 6 h have been studied on the basis of immunological recognition and chromatographic elution patterns. Overall concentrations of ERC and ERN did not change during this time period when receptor-saturating concentrations of [3H]E2 were maintained (Jakesz, R., Kasid, A., and Lippman, M. E. (1983) J. Biol. Chem. 258, 11798-11806); however, biochemical characteristics were different in ERC and ERN after short or long term hormonal exposure. When ERC from rats treated with estradiol for 30 min was applied to HAP or DEAE columns, two different ER binding components were seen. DNA binding in a cell-free system revealed that these binding components represented an activated and a nonactivated ERC population. After long term hormonal exposure (6 h), only one component of ERC with low DNA binding could be shown despite the preservation of an equivalent quantity of cytoplasmic binding activity. This binder does not react with a monoclonal antibody directed against extranuclear estrogen receptor species. These data suggest disappearance of the activated ERC population, with appearance of a new, immunologically nonrecognizable ERC species with 6 h of continuous hormonal exposure. Elution profiles of ERN on HAP chromatography reveal 2 different binding components at 30 min and at 6 h of continuous [3H]E2 exposure. There is an increase of the population eluted at higher molarity after 6 h of in vivo treatment. This later eluting binding component is the major DNA binder in vitro. ERN from both time points are recognized immunologically by monoclonal antibody. After reaction with the antibody, the sedimentation coefficient shifted to 8-9 S on sucrose gradients, but the previously described faster sedimentation of ERN extracted 6 h after injection persisted. We conclude that ER in both cellular compartments undergoes time-dependent alterations, which may be involved in the initiation of hormone action.