mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Lipid peroxidation in membranes and low-density lipoproteins: similarities and differences

Lipid peroxidation has been a central aspect of studies of the nature of free radical species and their origin in biological systems. Moreover, there has been a growing interest in lipid peroxidation based on evidence that biologically active products are formed that influence cell function and the course of major human diseases. A review of the work in this area is contributed by Lars Ernster is presented with an emphasis on the mechanisms by which lipid peroxidation is initiated in biological lipid systems. Based on what was described for metal catalyzed oxidation of cell membranes, and the seminal studies on cytochrome P-450-mediated lipid peroxidation, several parallel and distinct aspects of lipid peroxidation are described. A key distinction between lipid peroxidation in cell membranes and lipoproteins reveals aspects of free radical initiated reactions involving proteins and lipids that determine pro- vs. anti-oxidant outcomes, and the role of lipid structure and order in delineating the progress of oxidation.     

Changes in cellular gene expression in rat thyroid epithelial cells transformed by the Kirsten murine sarcoma virus

We have exploited a recently characterized system of rat thyroid epithelial cells transformed by the wild-type (wt) and a temperature-sensitive (ts) mutant strain of the Kirsten murine sarcoma virus (Ki-MSV) in order to study the effects of the K-ras oncogene on the gene expression of differentiated thyroid epithelial cells. By using cDNAs isolated from normal thyroid glands as probes, we were able to identify three sets of cellular sequences whose expression is influenced by the v-K-ras oncogene. The first set of genes is irreversibly repressed by transformation with both the wt and the ts viruses. The second set of genes is repressed in the ts-Ki-MSV-transformed cells but not in the same cells grown at the nonpermissive temperature. A third set of genes is present at higher levels at the nonpermissive temperature than at the permissive temperature. This system has allowed us to isolate and characterize a number of cDNA clones belonging to each of these three sets of genes. These specific cDNAs are suitable probes to study phenotypical changes during transformation of epithelial cells.     

Simultaneous 280 MHz EPR imaging of rat organs during nitroxide free radical clearance.

A radio frequency (RF) (280 MHz) electron paramagnetic resonance (EPR) spectroscopy and imaging apparatus has been used to localize a pyrrolidine nitroxide free radical in the rat abdomen and thorax. The nitroxide 2,2.5.5,-tetramethylpyrrolidine-1-oxyl-3- carboxylic acid (PCA) had a whole body monoexponential decay with half-life of 13.3 +/- 0.7 (n = 4), 19.4 +/- 0.2 (n = 3), and 23 +/- 2 (n = 6) min for 1, 2, and 3 mmol/kg PCA, respectively. Up to seven one-dimensional longitudinal projections were collected on six rats in the presence of a 8 mT/m field gradient. With an injection dose of 3 mmol/kg, PCA half-lives were 19 +/- 1, 17 +/- 2, and 22 +/- 2 min (n = 6) in the lower abdomen, in the liver, and in the thorax, respectively. Thorax half-life was significantly longer than liver half-life. Sequential two-dimensional images of PCA distribution in a plane longitudinal to the rat body were obtained from eight spectra in the presence of a gradient of 12 mT/m (acquisition time 5 min; spatial resolution 8 mm). After 7 min, the nitroxide was detectable in the left side of the thorax area, but it was mostly localized in the liver. PCA was more uniformly distributed in the image collected after 17 min.     

BDNF rs6265 methylation and genotype interact on risk for schizophrenia

Epigenetic mechanisms can mediate gene-environment interactions relevant for complex disorders. The BDNF gene is crucial for development and brain plasticity, is sensitive to environmental stressors, such as hypoxia, and harbors the functional SNP rs6265 (Val⁶⁶Met), which creates or abolishes a CpG dinucleotide for DNA methylation. We found that methylation at the BDNF rs6265 Val allele in peripheral blood of healthy subjects is associated with hypoxia-related early life events (hOCs) and intermediate phenotypes for schizophrenia in a distinctive manner, depending on rs6265 genotype: in ValVal individuals increased methylation is associated with exposure to hOCs and impaired working memory (WM) accuracy, while the opposite is true for ValMet subjects. Also, rs6265 methylation and hOCs interact in modulating WM-related prefrontal activity, another intermediate phenotype for schizophrenia, with an analogous opposite direction in the 2 genotypes. Consistently, rs6265 methylation has a different association with schizophrenia risk in ValVals and ValMets. The relationships of methylation with BDNF levels and of genotype with BHLHB2 binding likely contribute to these opposite effects of methylation. We conclude that BDNF rs6265 methylation interacts with genotype to bridge early environmental exposures to adult phenotypes, relevant for schizophrenia. The study of epigenetic changes in regions containing genetic variation relevant for human diseases may have beneficial implications for the understanding of how genes are actually translated into phenotypes.     

Mammalian small stress proteins protect against oxidative stress through their ability to increase glucose-6-phosphate dehydrogenase activity and by maintaining optimal cellular detoxifying machinery

The protective activity of small stress proteins (sHsp) against H2O2-mediated cell death in the highly sensitive murine L929 fibroblast has been analyzed. We report here that the human Hsp27- and murine Hsp25-mediated rise in glutathione (GSH) levels as well as the maintenance of this redox modulator in its reduced form was directly responsible for the protection observed at the level of cell morphology and mitochondrial membrane potential. sHsp expression also buffered the increase in protein oxidation following H2O2 treatment and protected several key enzymes against inactivation. In this case, however, the protection necessitated both an increase in GSH and the presence of sHsp per se since the pattern of protection against protein oxidation mediated by a simple GSH increase was different from that induced by sHsp expression. Among the enzymes analyzed, we noticed that sHsp significantly increased glucose-6-phosphate dehydrogenase (G6PD) activity and to a lesser extent glutathione reductase and glutathione transferase activities. Moreover, an increased GSH level was observed in G6PD-overexpressing L929 cell clones. Taken together our results suggest that sHsp protect against oxidative stress through a G6PD-dependent ability to increase and uphold GSH in its reduced form and by using this redox modulator as an essential parameter of their in vivo chaperone activity against oxidized proteins.     

Oxidative stress,spermatogenesis and fertility

Reactive oxygen species production and glutathione depletion in mammalian male germ cells are physiological events that are requisite to the functional maturation and capacitation of spermatozoa. In relation to this oxidative stress, an oxidation of the bulk of protein sulfydryl groups takes place during the final phases of male germ cell maturation. The selenoenzyme phospholipid hydroperoxide glutathione peroxidase catalyzes this reaction, and accounts for both the assembly of the mid-piece of spermatozoa and chromatin condensation. This process highlights the role of H2O2 and selenium in spermatogenesis and provides a mechanism for coupling a 'physiologically controlled' oxidative stress to a specialized phenotypic function.     

Male fertility is linked to the selenoprotein phospholipid hydroperoxide glutathione peroxidase

The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) accounts for almost the entire selenium content of mammalian testis. PHGPx is abundantly expressed in spermatids as active peroxidase but is transformed to an oxidatively inactivated protein in mature sperm, where it is a major constituent of the mitochondrial capsule in the midpiece. Male infertility in selenium-deficient animals, which is characterized by impaired sperm motility and morphological midpiece alterations, is considered to result from insufficient PHGPx content. We studied the relationship between sperm PHGPx, measured as rescued activity, and human fertility. Sperm specimens from 75 infertile men and 37 controls were analyzed for fertility-related parameters according to World Health Organization criteria. The PHGPx protein content was estimated after reductive solubilization of the spermatozoa by measuring the rescued PHGPx activity. Rescued PHGPx activity of infertile men ranged significantly below that of controls (93.2 +/- 60.1 units/mg sperm protein vs. 187.5 +/- 55.3 units/mg) and was particularly low in oligoasthenozoospermic specimens (61.93 +/- 45.42 units/mg; P < 0.001 compared with controls and asthenozoospermic samples). Rescued PHGPx activity was correlated positively with viability, morphological integrity, and most profoundly forward motility (r = 0.35, 0.44, and 0.45, respectively). In isolated motile samples, motility decreased faster with decreasing PHGPx content. In humans, PHGPx appears to be indispensable for structural integrity of spermatozoa and to codetermine sperm motility and viability. Because the content of PHGPx, irrespective of the cause of alteration, is correlated with fertility-related parameters, PHGPx can be considered a predictive measure for fertilization capacity.