Systemic Treatment with Erythropoietin Protects the Neurovascular Unit in a Rat Model of Retinal Neurodegeneration

Rats expressing a transgenic polycystic kidney disease (PKD) gene develop photoreceptor degeneration and subsequent vasoregression, as well as activation of retinal microglia and macroglia. To target the whole neuroglialvascular unit, neuro- and vasoprotective Erythropoietin (EPO) was intraperitoneally injected into four –week old male heterozygous PKD rats three times a week at a dose of 256 IU/kg body weight. For comparison EPO-like peptide, lacking unwanted side effects of EPO treatment, was given five times a week at a dose of 10 µg/kg body weight. Matched EPO treated Sprague Dawley and water-injected PKD rats were held as controls. After four weeks of treatment the animals were sacrificed and analysis of the neurovascular morphology, glial cell activity and pAkt localization was performed. The number of endothelial cells and pericytes did not change after treatment with EPO or EPO-like peptide. There was a nonsignificant reduction of migrating pericytes by 23% and 49%, respectively. Formation of acellular capillaries was significantly reduced by 49% (p<0.001) or 40% (p<0.05). EPO-treatment protected against thinning of the central retina by 10% (p<0.05), a composite of an increase of the outer nuclear layer by 12% (p<0.01) and in the outer segments of photoreceptors by 26% (p<0.001). Quantification of cell nuclei revealed no difference. Microglial activity, shown by gene expression of CD74, decreased by 67% (p<0.01) after EPO and 36% (n.s.) after EPO-like peptide treatment. In conclusion, EPO safeguards the neuroglialvascular unit in a model of retinal neurodegeneration and secondary vasoregression. This finding strengthens EPO in its protective capability for the whole neuroglialvascular unit.     

World-wide survey of an Accord insertion and its association with DDT resistance in Drosophila melanogaster

Previous work showed that insecticide resistance in Drosophila melanogaster is correlated with the insertion of an Accord-like element into the 5' region of the cytochrome P450 gene, Cyp6g1. Here, we study the distribution of the Accord-like element in 673 recently collected D. melanogaster lines from 34 world-wide populations. We also examine the extent of microsatellite variability along a 180-kilobase (kb) genomic region of chromosome II encompassing the resistance gene. We confirm a 100% correlation of the Accord insertion with insecticide resistance and a significant reduction in variability extending at least 20 kb downstream of the Cyp6g1 gene. The frequency of the Accord insertion differs significantly between East African (32-55%) and nonAfrican (85-100%) populations. This pattern is consistent with a selective sweep driving the Accord insertion close to fixation in nonAfrican populations as a result of the insecticide resistance phenotype it confers. This study confirms that hitchhiking mapping can be used to identify beneficial mutations in natural populations.     

Polymorphism and Locus-Specific Effects on Polymorphism at Microsatellite Loci in Natural Drosophila Melanogaster Populations

We have studied the natural variation at microsatellite loci in two African and five non-African populations of Drosophila melanogaster. Ten dinucleotide simple sequence loci were cloned from chromosomally mapped P1 clones and typed for single individuals from isofemale lines of the respective populations. We find that the African populations harbor the largest degree of diversity, while the non-African populations show a lower diversity. This supports previous results that D. melanogaster originated in Africa and spread across the rest of the world in historic times. Using genetic distance measures, we find also a distinct population subdivision between the non-African populations. Most interestingly, we find for some loci in some populations a strongly reduced variability, which cannot be explained by bottleneck effects. Employing a conservative test based on the variance in repeat number, we find that at least one locus in one population deviates significantly from the expectations of mutation-drift equilibrium. We suggest that this may be due to a recent selective sweep in this chromosomal region that may have been caused by a linked locus that was involved in local adaptation of the population.     

Limb vasodilation provoked by stimulation of the carotid sinus. Importance of histaminergy in the dog

The vasodilator reflex induced by baroreceptor stimulation was studied on the hindlimbs of the dog. The reflex was induced by norepinephrine (1 microgram/kg) either by intravenous injection or by direct injection into the carotid sinus. In other experiences, the baroreceptor stimulation was obtained by distension of the sinus by rapid injection of 100 ml of physiological serum. The vascular response was studied by recording the hindlimbs blood flow. One of the limbs was previously pretreated by mepyramine and cimetidine (blockage of histaminergic H1 and H2 receptors). During the first minute after the baroreceptor stimulation, blood samples were collected from the venous blood of hindlimbs for histamine assay (fluorometric assay). Our results show: a much lower vasodilation on the limb pretreated by histamine antagonist, a significant increase during the reflex vasodilation of histamine blood levels measured in the efferent blood of hindlimbs. These results, obtained in experimental conditions as physiological as possible (blood perfusion of the limbs with "natural" hemodynamic parameters) permit to conclude that the vasodilation induced by baroreceptor reflex is at least partially histaminergic in the dog.     

Apurinic/apyrimidinic endonuclease 1, p53, and thioredoxin are linked in control of aging in C. elegans

Deletions in mitochondrial DNA (mtDNA) accumulate during aging. Expression of the Caenorhabditis elegans apurinic/apyrimidinic endonuclease 1 (APE1) ortholog exo‐3, involved in DNA repair, is reduced by 45% (P < 0.05) during aging of C. elegans. Suppression of exo‐3 by treatment with RNAi resulted in a threefold increase in mtDNA deletions (P < 0.05), twofold enhanced generation of reactive oxygen species (ROS) (P < 0.01), distortion of the structural integrity of the nervous system, reduction of head motility by 43% (P < 0.01) and whole animal motility by 38% (P < 0.05). Suppression of exo‐3 significantly reduced life span: mean life span decreased from 18.5 ± 0.4 to 15.4 ± 0.1 days (P < 0.001) and maximum life span from 25.9 ± 0.4 to 23.2 ± 0.1 days (P = 0.001). Additional treatment of exo‐3‐suppressed animals with a mitochondrial uncoupler decreased ROS levels, reduced neuronal damage, and increased motility and life span. Additional suppression of the C. elegans p53 ortholog cep‐1 in exo‐3 RNAi‐treated animals similarly decreased ROS levels, preserved neuronal integrity, and increased motility and life span. In wild‐type animals, suppression of cep‐1, involved in downregulation of exo‐3, increased expression of exo‐3 without a significant effect on ROS levels, preserved neuronal integrity, and increased motility and life span. Suppression of the C. elegans thioredoxin orthologs trx‐1 and trx‐2, involved in the redox chaperone activity of exo‐3, overrides the protective effect of cep‐1 RNAi treatment on neuronal integrity, neuronal function, mean and maximum life span. These results show that APE1/EXO‐3, p53/CEP‐1, and thioredoxin affect each other and that these interactions determine aging as well as neuronal structure and function.     

Comparative evolutionary analysis of rDNA ITS regions in Drosophila

The internal transcribed spacer (ITS) of the ribosomal DNA is generally considered to be under low functional constraint, and it is therefore often treated as a typical nonfunctional spacer sequence. We have analyzed the ITS regions of five species from the Drosophila melanogaster subgroup, two Drosophila species from outside this group (D. pseudoobscura and D. virilis), as well as from the more distantly related dipteran fly Musca domestica. The sequence comparisons show a distinctive conservation/divergence pattern, indicating that some regions are more conserved than others. Moreover, secondary-structure calculations indicate several conserved structural elements within the ITS regions. On the other hand, a statistical test that allows us to estimate the fraction of sites that are not under selective constraint suggests that more than half of the spacer is apparently free to diverge and evolves with a rate that is close to the neutral rate of sequence evolution in Drosophila. The ITS sequences can be used to derive a molecular phylogeny for the species under study. We find that the ITS tree is largely in line with the so-far-known phylogeny of this group of species, with one difference. The species most distant within the D. melanogaster subgroup is D. yakuba, rather than D. orena, as is normally assumed.