The X4 phenotype of HIV type 1 evolves from R5 in two children of mothers, carrying X4, and is not linked to transmission.

Previously, we found that emergence of the X4 viral phenotype in HIV-1-infected children was related to the presence of X4 in their mothers (C.H. Casper et al., J Infect Dis 2002; 186:914-921). Here, we investigated the origin of the X4 phenotype in the child, analyzing two mother-child pairs (Ma-Ca, Mb-Cb) where the mothers carried X4 and their children developed X4 after an initial presence of R5. We used nested polymerase chain reaction of the env V3 region to generate 203 HIV-1 clones for sequencing (Ma, n = 44; Ca, n = 73; Mb, n = 61; Cb, n = 25) from DNA of peripheral blood mononuclear cell (PBMC) lysates, altogether 167 clones, or from cDNA of plasma RNA, 36 clones. PBMC and plasma isolate sequences from each time point enabled us to assign the probable phenotype to clone sequences in a phylogenetic tree. The transmission and evolution were reconstructed using the maximum likelihood method. In mother-child pair Ma-Ca, one maternal R5 isolate clustered with the child's R5 sequences, at the earliest time when R5 was isolated in the child, confirming this as a likely source of the transmitted R5 phenotype. At age 3, an X4 population was present in the child that had evolved from the child's own R5-associated population, clearly distinct from the maternal X4 sequences. The second mother-child pair (Mb-Cb) displayed a similar pattern. Amino acid substitution patterns corroborated the conclusions from the phylogenetic tree. Thus, in both children, the X4 virus developed from their own R5 population, and was not caused by transmission of X4.

Binding of dietary cobalt to sarcoplasmic reticulum proteins.

It has previously been shown that cobalt accumulates in the myocardium of rats, mainly the sarcoplasmic reticulum (SR) and the mitochondrial inner membrane. In order to investigate the mode of accumulation of cobalt in the SR, rats were given a dietary cobalt supplementation of 40 mg of CoSO4 x 7H2O kg-1 body wt, after which the rats were sacrificed and the sarcoplasmic reticulum was isolated. The SR proteins were subjected to analysis by polyacrylamide gel electrophoresis followed by protein staining and determination of the content of cobalt in each protein band. The major cobalt-binding protein was found to have a molecular weight of about 100,000; a 200,000 molecular weight protein was also found to bind cobalt, although less extensively. These results suggest that cobalt is bound to the monomeric and dimeric forms of Ca(2+)-ATPase in the SR of the myocardium.

Energy-linked transhydrogenase. Effects of valinomycin and nigericin on the ATP-driven transhydrogenase reaction catalyzed by reconstituted transhydrogenase-ATPase vesicles.

Reconstituted transhydrogenase-ATPase vesicles obtained with purified beef heart transhydrogenase and oligomycin-sensitive ATPase were investigated with respect to the mode of interaction between the two proton pumps, with special reference to the relative contributions of the membrane potential and proton gradient using valinomycin and nigericin in the presence of potassium. In the absence of ionophores and at low ATP concentrations, below 20 microM, the ATPase generated a proton motive force which was predominantly due to a membrane potential, whereas at saturating concentrations of ATP the proton gradient was the predominant component. The ATP-dependence of the rate of the ATP-driven transhydrogenase reaction showed apparent Km values in the low and high ATP concentration range of about 3 and 56 microM, respectively, with a corresponding difference in Vmax of about 3-fold. It is concluded that the reconstituted transhydrogenase can utilize both a membrane potential and a proton gradient, separately or combined, where the relative contributions of these components depend on the activity of the ATPase. In the reconstituted vesicles, the maximally active transhydrogenase is apparently driven by an electrochemical proton gradient where the membrane potential and the proton gradient contribute one-third and two-thirds, respectively. The rate-dependent relative generation of a membrane potential and pH gradient presumably reflects the proton pump characteristics of the ATPase and/or buffering/permeability characteristics of the vesicles rather than the properties of the transhydrogenase per se. These results are discussed in relation to current models for transhydrogenase-linked proton translocation.

On the presence of a nicotinamide nucleotide transhydrogenase in mitochondria from potato tuber.

Mitochondria isolated from potato (Solanum tuberosum L.) tuber were investigated for the presence of a nicotinamide nucleotide transhydrogenase activity. Submitochondrial particles derived from these mitochondria by sonication catalyzed a reduction of NAD(+) or 3-acetylpyridine-NAD(+) by NADPH, which showed a maximum of about 50 to 150 nanomoles/minute.milligram protein at pH 5 to 6. The K(m) values for 3-acetylpyridine-NAD(+) and NADPH were about 24 and 55 micromolar, respectively. Intact mitochondria showed a negligible activity in the absence of detergents. However, in the presence of detergents the specific activity approached about 30% of that seen with submitochondrial particles. The potato mitochondria transhydrogenase activity was sensitive to trypsin and phenylarsine oxide, both agents that are known to inhibit the mammalian transhydrogenase. Antibodies raised against rat liver transhydrogenase crossreacted with two peptides in potato tuber mitochondrial membranes with a molecular mass of 100 to 115 kilodaltons. The observed transhydrogenase activities may be due to an unspecific activity of dehydrogenases and/or to a genuine transhydrogenase. The activity contributions by NADH dehydrogenases and transhydrogenase to the total transhydrogenase activity were investigated by determining their relative sensitivities to trypsin. It is concluded that, at high or neutral pH, the observed transhydrogenase activity in potato tuber submitochondrial particles is due to the presence of a genuine and specific high molecular weight transhydrogenase. At low pH an unspecific reaction of an NADH dehydrogenase with NADPH contributes to the total trans-hydrogenase activity.

Affinity chromatography of mitochondrial nicotinamide nucleotide transhydrogenase.

The binding of mitochondrial nicotinamide nucleotide transhydrogenase to NAD+ and NADP+ immobilized to agarose through different parts of the nicotinamide nucleotide molecule was investigated. NADP+ bound at the C8 atom in the adenine moiety proved to be the most efficient ligand whereas that bound at the C3 atom of the ribose moiety was relatively inefficient. NAD+ ligands were generally inactive independently of the site of attachment. Previous results suggest, however, that binding to immobilized NAD+ may be influenced by the detergent in which transhydrogenase is dispersed. Binding to neither ligand was affected by the presence of the second substrate.

Cell-free translation of mitochondrial nicotinamide nucleotide transhydrogenase.

Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc1 complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.