Activity of branched-chain 2-oxo acid dehydrogenase complex in rat liver mitochondria and in rat liver.

Four mitochondrial marker enzymes were used to show that: (1) high-protein (24%) diet increased the rat liver concentration and content of total branched-chain 2-oxo acid dehydrogenase complex (BCDC) by 31% by increasing mitochondrial specific activity of BCDC; (2) starvation increased the liver concentration of BCDC by 25% by decreasing liver weight; the liver content of mitochondria and the mitochondrial specific activity of BCDC were unchanged; (3) protein-free diet decreased rat liver BCDC concentration and content by 20%, by decreasing the liver concentration and content of mitochondria. Protein-free diet increased liver mitochondrial specific activities of L-glutamate, 2-oxoglutarate and NAD-isocitrate dehydrogenases. The validity of a mitochondrial method for the determination of the liver concentration of BCDC and the percentage in the active form in vivo is confirmed, and improvements are described. The experimental basis of criticisms of its use in this regard by Zhang, Paxton, Goodwin, Shimomura & Harris [(1987) Biochem. J. 246, 625-631] was not confirmed. The finding by Harris, Powell, Paxton, Gillim & Nagae [(1985) Arch. Biochem. Biophys. 243, 542-555], that starvation has no effect on the percentage of BCDC in the active form in rat liver, is confirmed.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Dephosphorylation and reactivation of phosphorylated purified ox-kidney branched-chain dehydrogenase complex by co-purified phosphatase

The branched-chain 2 oxoacid dehydrogenase complex has been purified from well-washed ox-kidney mitochondria together with branched-chain dehydrogenase kinase. The complex was inactivated and phosphorylated by ATP in about 5 min at 30 degrees C. After hydrolysis of ATP by a contaminating ATPase (5-10 min) the complex was dephosphorylated and reactivated. Dephosphorylation and reactivation were linearly correlated. Reactivation was dependent upon Mg2+ (K0.5 greater than 1 mM) and inhibited completely by 50 mM fluoride. Reactivation and dephosphorylation are attributed to a mitochondrial branched-chain dehydrogenase phosphatase.     

Perifused adenohypophyseal cells: release of ACTH and kinetics of the response to secretagogues

The release of ACTH-like immunoactivity (ACTH-LI) from perifused dispersed adenohypophyseal cells was examined under basal conditions and in response to various secretagogues. Frequent sampling of effluent perfusion medium and a variable stimulation format allowed us to discern differences in the effects of the secretagogues. The dye, dextran blue, was used to define the kinetics of flow intrinsic to the perifusion system, allowing a detailed analysis of the responses to secretagogues. Each agent had a time course of action which could be related to its supposed site and mode of action in these cells. A crude hypothalamic extract, a partially purified corticotrophin releasing factor preparation, 8-bromoadenosine 3',5'-cyclic monophosphate (8Br-cAMP), and 3-isobutyl-1-methylxanthine all caused dose-related, repeatable increases of the release of ACTH-LI. A 10-fold elevation in the concentration of K+ in the perifusion medium (10K) caused a transient increase in the release of ACTH-LI which was reduced when repeated. These results suggest that 10K stimulates the release of ACTH-LI only from a "readily-releasable pool." The other agents appear to affect the release process more profoundly, for example, by stimulating intracellular transport of the ACTH-LI.     

Twin studies demonstrate a host cell genetic effect on productive human immunodeficiency virus infection of human monocytes and macrophages in vitro.

Biological and genetic variability is a prominent feature of human immunodeficiency virus (HIV) strains, especially in tropism, syncytium formation, and replicative capacity. To determine whether there were variable host cell effects on HIV replication in monocytes, three different strains of low-passage-number monocytotropic blood isolates of HIV and the laboratory-adapted strain Ba-L were inoculated into panels of adherent monocytes drawn from 44 different donors, and peak extracellular HIV p24 antigen titers were compared. The clinical HIV strains showed patterns of either moderate or low-level replication in most donor monocytes (20 to 4,000 pg/ml). However, within this range there was marked variation in peak titers in most donors. HIV type 1 Ba-L replicated in all donor monocytes to much higher levels with less variability (30 to 40 ng/ml). Furthermore, replication of 21 clinical blood-derived strains of HIV in blood monocytes and monocyte-derived macrophages (MDM) from pairs of identical twins and age-matched unrelated donors (URD) of the same sex were compared. In all of the seven pairs of identical twins, the kinetics of replication (measured by extracellular HIV p24 antigen) of panels of four clinical HIV type 1 isolates in monocytes were similar within pairs. However, marked and significant differences in kinetics of HIV production occurred within 10 of the 12 unrelated donor pairs (P = 0.0007). The remaining two URD pairs showed similar kinetic patterns, but only one pair had the same HLA-DR genotype. Similar results were observed with monocytes/MDMs obtained from a second bleed of the same donor. Hence, discordant patterns of HIV replication kinetics between URD monocyte pairs contrasted with concordant patterns in identical twin monocytes. These data strongly suggest a host cell genetic effect on productive viral replication in monocytes and MDMs. So far, no consistent genetic linkage of HIV replication pattern with HLA-DR genotype has been observed.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.