Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Various properties of purified hepatoredoxin from bovine liver mitochondria

Hepatoredoxin purified to homogeneity from bovine liver mitochondria has been characterized for the first time in terms of its most important physico-chemical properties. The protein was found to contain in its active center a [2Fe-2S] cluster and has in the oxidized state an absorption maxima at 280, 320, 415 and 455 nm. The spectrophotometric index of purity, A415/A280 of the homogeneous native preparation is 0.84; extinction coefficient, epsilon 415, is 9800 M-1cm-1. The Mr of hepatoredoxin as evidenced by data from SDS gel electrophoresis is 12 500 Da; pI is 4.2. Hepatoredoxin is necessary for the reconstitution of the C27-steroid hydroxylase activity and can be substituted for by a related protein, adrenodoxin. All the above parameters as well as the circular dichroism spectra, immunochemical properties and sequence of the initial five N-terminal amino acids of hepatoredoxin and adrenodoxin are either coincident or very close. At the same time, the amino acid composition of these ferredoxins, apart from some common features, has individual peculiarities.     

Concentration of T3 and T4 in blood of non-irradiated and irradiated with different doses rats fasted for two days beforehand

There were no changes in concentration of T3 and T4 in blood of the rats that were irradiated with a dose of 0.5, 1, 2, 4 and 6 Gy in comparison with non-irradiated rats, if the animals were not fed for two days before decapitation. This suggests that the effect of ionizing radiation on thyroid function is mediated by anorexia syndrome. The decrease in concentration of T4 after exposure to 8 Gy cannot be explained by postradiative anorexia and most likely is connected with starting enterotoxemy in difficult cases of acute radiation sickness.     

Insulin function in rats at early terms after 4 Gy whole-body irradiation

In the present study we made an attempt to estimate changes of insulin function at early terms after external irradiation of rats. Experimental conditions: male albino rats were studied 7; 14; 21; 28 days after the external whole-body gamma-irradiation (137Cs; 4 Gy). For this purpose the kinetics of 125I-insulin disappearance from blood plasma was investigated. Simultaneously dynamics of insulin blood concentration was studied in practically full and fasting animals. On the basis of the data received the following basic pharmacokinetic parameters were designed according to the two-compartmental model: central and peripheral compartment volumes, transfer and elimination rates, turnover and metabolic clearance rates. No substantial changes in insulin clearance were found compared to controls in all the postirradiation terms investigated. Hence, the changes in the turnover rate of insulin are proportional to blood hormone concentration. The significant increase of concentration and turnover was observed only 7 days after irradiation in rats with free access to food. The data received suggest that the insulin function of a pancreas in an organism exposed to a 4 Gy dose is maintained at a level sufficient for ensuring adequate regulation of the glucose homeostasis and of the carbohydrate metabolism.     

Finding functional sites in structural genomics proteins

Assigning function to structures is an important aspect of structural genomics projects, since they frequently provide structures for uncharacterized proteins. Similarities uncovered by structure alignment can suggest a similar function, even in the absence of sequence similarity. For proteins adopting novel folds or those with many functions, this strategy can fail, but functional clues can still come from comparison of local functional sites involving a few key residues. Here we assess the general applicability of functional site comparison through the study of 157 proteins solved by structural genomics initiatives. For 17, the method bolsters confidence in predictions made based on overall fold similarity. For another 12 with new folds, it suggests functions, including a putative phosphotyrosine binding site in the Archaeal protein Mth1187 and an active site for a ribose isomerase. The approach is applied weekly to all new structures, providing a resource for those interested in using structure to infer function.     

A radioimmunoprecipitation method in the serodiagnosis of HIV infection: the development of the optimal conditions for its performance and the evaluation of the possibilities for its use

The optimum conditions for using the method of radioimmunoprecipitation (RIP) for the detection of human immunodeficiency virus (HIV) in serum samples have been established. Out of several available cell lines persistently infected with HIV, specially selected line 17 has been chosen. The characteristic feature of this is the high and stable (under the conditions of prolonged cultivation) accumulation of virus-specific proteins in infected cells. The optimum conditions for making the test and its evaluation have also been established. The data of literature on the advantages of the method of RIP over such traditional methods as the enzyme immunoassay and immunoblotting have been confirmed. Thus, the presence of specific antibodies in several serum samples registered as false negative has been established. The intertypical reactivity of two serotypes of the virus, HIV-1 and HIV-2, has been studied. Cross reactivity of antibodies with respect to the HIV gene gag, but not with respect to viral glycoproteids, has been established. Ideas on the expediency and prospects of using RIP for the serological control of HIV infection are presented.     

The covalent-sorption reconstitution of steroid hydroxylases

The effect of covalent immobilization via free amino groups on the catalytic activity of individual components of the cholesterol side-chain cleavage and 11b-steroid hydroxylation systems (adrenodoxin reductase, adrenodoxin, cytochrome P-450scc and cytochrome P-450(11)b) as well as on that of co-immobilized protein complexes. The protein complex formation at different stages of the monooxygenase cycle (i.e., reduction, oxygenation) was followed by direct spectrophotometric monitoring of the functional state of the immobilized complexes. Cholesterol side-chain cleavage was carried out in minicolumns, using various combinations of immobilized and soluble proteins. Cytochromes P-450scc and P-450(11)b were found to retain their functional activities after immobilization via free SH-groups.