Voltage dependence of Na-Ca exchanger conformational currents.

Properties of a transient current (Icont) believed to reflect a conformational change of the Na-Ca exchanger molecules after Ca2+ binding were investigated. Intracellular Ca2+ concentration jumps in isolated cardiac myocytes were generated with flash photolysis of caged Ca2+ dimethoxynitrophenamine, and membrane currents were simultaneously measured using the whole-cell variant of the patch-clamp technique. A previously unresolved shallow voltage dependence of Icont was revealed after developing an experimental protocol designed to compensate for the photoconsumption of the caged compound. This voltage dependence can be interpreted to reflect the distribution of Na-Ca exchanger conformational states with the Ca2+ binding site exposed to the inside of the cell immediately before the flash. Analysis performed by fitting a Boltzmann distribution to the observed data suggests that under control conditions most exchanger molecules reside in states with the Ca2+ binding site facing the outside of the cell. Dialysis of the cytosol with 3',4'-dichlorobenzamil, an organic inhibitor of the Na-Ca exchange, increased the magnitude of Icont and changed the voltage dependence, consistent with a parallel shift of the charge/voltage curve. This shift may result from intracellular DCB interfering with an Na(+)-binding or Na(+)-translocating step. These observations are consistent with Icont arising from a charge movement mediated by the Na-Ca exchanger molecules after binding of Ca2+.     

Epstein-Barr virus transformation of human B lymphocytes despite inhibition of viral polymerase.

Epstein-Barr virus transformed human lymphocytes despite the presence of up to 500 microM acyclovir [9-(2-hydroxyethoxymethyl)guanine], a viral DNA polymerase inhibitor. The transformed cells contained multiple Epstein-Barr virus genome copy numbers. Functional viral DNA polymerase is probably not required for cell transformation and the initial amplification of the viral genome.     

Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.     

Heterozygote advantage in Tay-Sachs carriers?

Chi-square analyses of new data as well as data previously reported by Myrianthopoulos have shown that grandparents of Tay-Sachs carriers die from proportionally the same causes as grandparents of noncarriers. It is unlikely that there is any advantage to being a Tay-Sachs carrier insofar as resistance to tuberculosis is concerned. Our results are further evidence to support Fraikor's claim that the high carrier frequency of the allele in Ashkenazi Jews is probably caused by a combination of founder effect, genetic drift, and differential immigration patterns.     

Inbreeding reveals interpopulation differences in inversion polymorphism of Drosophila subobscura

The adaptive significance of inversion polymorphism of Drosophila subobscura is well established. However, little is known about gene combinations within inversions which are coadapted because of population-specific effects. We studied this aspect of Dobzhansky's coadaptation hypothesis, using the systematic inbreeding method. Differences in magnitude and quality of inversion polymorphism in two ecologically and topologically distinct habitats were compared after several generations of continuous full-sib inbreeding. Populations from the two habitats differ in frequency of homokaryotypes after third and fifth generation of inbreeding and in the levels of homozygosity of different gene arrangements. The effect of homozygosity appears population and chromosome specific.     

Carotenoid synthesis in Streptomyces setonii ISP5395 is induced by the gene crtS, whose product is similar to a sigma factor

In many species of actinomycetes, carotenogenesis can be photoinduced. The capacity to respond to photoinduction is, however unstable and, in various strains of Streptomyces, is lost at a relatively high frequency. In Streptomyces setonii ISP5395, which normally produces no carotenoids, carotenoid-producing mutants can be obtained following protoplast regeneration. We report here the characterization of a gene, crtS, which was isolated from one such mutant and can confer on wild-type S. setonii ISP5395 cells the capacity to synthesize carotenoids. Sequence analysis of crtS reveals an open reading frame, which shows homology to genes that encode alternative sigma factors in Bacillus subtilis. We propose that crtS encodes a sigma factor which is necessary for the expression of a cryptic gene(s) for carotenoid biosynthesis in S. setonii ISP5395.     

Determination of oxalyl thiolesters, N-oxalylcysteine and N-oxalylcysteamine in biological materials

A convenient method is described for the quantitative analysis of oxalyl thiolesters (OTEs), a newly discovered class of mammalian metabolites, in biological samples. By this particular technique the total concentration of all OTEs in the sample is determined. The method involves first reacting the biological material with cysteamine (2-aminoethanethiol) or cysteine under conditions that convert OTEs quantitatively to N-oxalylcysteamine (or N-oxalylcysteine), followed by reaction with monobromobimane to give a highly fluorescent derivative that is analyzed by reversed-phase ion-pair chromatography, with tetrabutylammonium ion as the counterion and N-(2-mercaptopropionyl)glycine as an internal standard. The method is capable of detecting as little as 0.6 pmol of the bimane derivative of the N-oxalyl compound in a single HPLC injection. The application of this method has led to the discovery that not only OTEs but also N-oxalylcysteine and N-oxalylcysteamine are normal mammalian metabolites. In various rat tissues the OTE concentration ranges up to 65 nmol/g (wet wt), the N-oxalylcysteine concentration is approximately 10 nmol/g, and the N-oxalylcysteamine concentration is 0-3 nmol/g.