Urocortin-1 within the centrally-projecting Edinger-Westphal nucleus is critical for ethanol preference

Converging lines of evidence point to the involvement of neurons of the centrally projecting Edinger-Westphal nucleus (EWcp) containing the neuropeptide Urocortin-1 (Ucn1) in excessive ethanol (EtOH) intake and EtOH sensitivity. Here, we expanded these previous findings by using a continuous-access, two-bottle choice drinking paradigm (3%, 6%, and 10% EtOH vs. tap water) to compare EtOH intake and EtOH preference in Ucn1 genetic knockout (KO) and wild-type (WT) mice. Based on previous studies demonstrating that electrolytic lesion of the EWcp attenuated EtOH intake and preference in high-drinking C57BL/6J mice, we also set out to determine whether EWcp lesion would differentially alter EtOH consumption in Ucn1 KO and WT mice. Finally, we implemented well-established place conditioning procedures in KO and WT mice to determine whether Ucn1 and the corticotropin-releasing factor type-2 receptor (CRF-R2) were involved in the rewarding and aversive effects of EtOH (2 g/kg, i.p.). Results from these studies revealed that (1) genetic deletion of Ucn1 dampened EtOH preference only in mice with an intact EWcp, but not in mice that received lesion of the EWcp, (2) lesion of the EWcp dampened EtOH intake in Ucn1 KO and WT mice, but dampened EtOH preference only in WT mice expressing Ucn1, and (3) genetic deletion of Ucn1 or CRF-R2 abolished the conditioned rewarding effects of EtOH, but deletion of Ucn1 had no effect on the conditioned aversive effects of EtOH. The current findings provide strong support for the hypothesis that EWcp-Ucn1 neurons play an important role in EtOH intake, preference, and reward.     

Antioxidant activity of fractionated extracts of rhizomes of high-altitude Podophyllum hexandrum: Role in radiation protection

Whole extract of rhizomes of Podophyllum hexandrum has been reported earlier by our group to render whole-body radioprotection. High-altitude P. hexandrum (HAPH) was therefore fractionated using solvents of varying polarity (non-polar to polar) and the different fractions were designated as, n-hexane (HE), chloroform (CE), alcohol (AE), hydro-alcohol (HA) and water (WE). The total polyphenolic content (mg% of quercetin) was determined spectrophotometrically, while. The major constituents present in each fraction were identified and characterized using LC-APCI/MS/MS. In vitro screening of the individual fractions, rich in polyphenols and lignans, revealed several bioactivities of direct relevance to radioprotection e.g. metal-chelation activity, antioxidant activity, DNA protection, inhibition of radiation (250 Gy) and iron/ascorbate-induced lipid peroxidation (LPO). CE exhibited maximum protection to plasmid (pBR322) DNA in the plasmid relaxation assay (68.09% of SC form retention). It also showed maximal metal chelation activity (41.59%), evaluated using 2,2-bipyridyl assay, followed by AE (31.25%), which exhibited maximum antioxidant potential (lowest absorption unit value: 0.0389± 0.00717) in the reducing power assay. AE also maximally inhibited iron/ascorbate-induced and radiation-induced LPO (99.76 and 92.249%, respectively, at 2000 g/ml) in mouse liver homogenate. Under conditions of combined stress (radiation (250 Gy) + iron/ascorbate), at a concentration of 2000 g/ml, HA exhibited higher percentage of inhibition (93.05%) of LPO activity. HA was found to be effective in significantly (p < 0.05) lowering LPO activity over a wide range of concentrations as compared to AE. The present comparative study indicated that alcoholic (AE) and hydro-alcoholic (HA) fractions are the most promising fractions, which can effectively tackle radiation-induced oxidative stress.     

Different population histories of the Mundari- and Mon-Khmer-speaking Austro-Asiatic tribes inferred from the mtDNA 9-bp deletion/insertion polymorphism in Indian populations

Length variation in the human mtDNA intergenic region between the cytochrome oxidase II (COII) and tRNA lysine (tRNA^lys) genes has been widely studied in world populations. Specifically, Austronesian populations of the Pacific and Austro-Asiatic populations of southeast Asia most frequently carry the 9-bp deletion in that region implying their shared common ancestry in haplogroup B. Furthermore, multiple independent origins of the 9-bp deletion at the background of other mtDNA haplogroups has been shown in populations of Africa, Europe, Australia, and India. We have analyzed 3293 Indian individuals belonging to 58 populations, representing different caste, tribal, and religious groups, for the length variation in the 9-bp motif. The 9-bp deletion (one copy) and insertion (three copies) alleles were observed in 2.51% (2.15% deletion and 0.36% insertion) of the individuals. The maximum frequency of the deletion (45.8%) was observed in the Nicobarese in association with the haplogroup B5a D-loop motif that is common throughout southeast Asia. The low polymorphism in the D-loop sequence of the Nicobarese B5a samples suggests their recent origin and a founder effect, probably involving migration from southeast Asia. Interestingly, none of the 302 (except one Munda sample, which has 9-bp insertion) from Mundari-speaking Austro-Asiatic populations from the Indian mainland showed the length polymorphism of the 9-bp motif, pointing either to their independent origin from the Mon-Khmeric-speaking Nicobarese or to an extensive admixture with neighboring Indo-European-speaking populations. Consistent with previous reports, the Indo-European and Dravidic populations of India showed low frequency of the 9-bp deletion/insertion. More than 18 independent origins of the deletion or insertion mutation could be inferred in the phylogenetic analysis of the D-loop sequences.     

Very long chain fatty acids activate NADPH oxidase in human dermal fibroblasts

Very long chain fatty acids (VLCFAs) are exclusively oxidized in peroxisomes and their levels are significantly increased in tissues of patients with peroxisomal disorders. Although the biochemical indicators of peroxisomal dysfunction, such as elevated VLCFAs, are well known, the mechanisms of pathogenesis of peroxisomal diseases are unclear. In this study we have examined the effect of VLCFAs on NADPH oxidase (NOX), a complex enzyme system responsible for the production of superoxide anions, in order to understand the oxidative stress-mediated mechanisms involved in pathology of peroxisomal disorders. Varying concentrations (2.5 to 10 microg ml(-1)) of VLCFAs, lignoceric acid and cerotic acid, significantly (p < 0.001) increased the enzymic activity of NOX in cultures of human dermal fibroblasts. VLCFAs did not affect the expression of gp91phox or p22phox whereas the mRNA and protein levels of p47phox were significantly (two or three-fold) increased following treatment of fibroblasts with lignoceric acid or cerotic acid. VLCFAs also caused a significant (p < 0.01) increase in lipid peroxidation in dermal fibroblasts which could be markedly reversed by treatment with apocyanin (10 mM) or superoxide dismutase (SOD, 25 U ml(-1)). With these results, we report for the first time that VLCFAs enhance NOX activity and superoxide anion-mediated lipid peroxidation in cultured dermal fibroblasts. This study proposes a mechanism that may be taking place in vivo during peroxisomal dysfunction and that leads to oxidative stress-mediated pathogenesis.     

Molecular epidemiology of simian immunodeficiency virus SIVsm in U.S. primate centers unravels the origin of SIVmac and SIVstm

Retrospective molecular epidemiology was performed on samples from four sooty mangabey (SM) colonies in the United States to characterize simian immunodeficiency virus SIVsm diversity in SMs and to trace virus circulation among different primate centers (PCs) over the past 30 years. The following SIVsm sequences were collected from different monkeys: 55 SIVsm isolates from the Tulane PC sampled between 1984 and 2004, 10 SIVsm isolates from the Yerkes PC sampled in 2002, 7 SIVsm isolates from the New Iberia PC sampled between 1979 and 1986, and 8 SIVsm isolates from the California PC sampled between 1975 and 1977. PCR and sequencing were done to characterize the gag, pol, and env gp36 genes. Phylogenetic analyses were correlated with the epidemiological data. Our analysis identified nine different divergent phylogenetic lineages that cocirculated in these four SM colonies in the Unites States in the past 30 years. Lineages 1 to 5 have been identified previously. Two of the newly identified SIVsm lineages found in SMs are ancestral to SIVmac251/SIVmac239/SIVmne and SIVstm. We further identified the origin of these two macaque viruses in SMs from the California National Primate Research Center. The diversity of SIVsm isolates in PCs in the United States mirrors that of human immunodeficiency virus type 1 (HIV-1) group M subtypes and offers a model for the molecular epidemiology of HIV and a new approach to vaccine testing. The cocirculation of divergent SIVsm strains in PCs resulted in founder effects, superinfections, and recombinations. This large array of SIVsm strains showing the same magnitude of diversity as HIV-1 group M subtypes should be extremely useful for modeling the efficacy of vaccination strategies under the real-world conditions of HIV-1 diversity. The genetic variability of SIVsm strains among PCs may influence the diagnosis and monitoring of SIVsm infection and, consequently, may bias the results of pathogenesis studies.     

Mutational study of the bacterial hemoglobin distal heme pocket

Ligand binding experiments on three mutants in the distal heme pocket of Vitreoscilla hemoglobin (GlnE7His, ProE8Ala, and GlnE7His,ProE8Ala) were used to probe the role of GlnE7 and ProE8 in the pocket's unusual structure. The oxygen dissociation constants for the wild type, E8Ala mutant, and E7His mutant proteins were 4.5, 4.7, and 1.7microM, respectively; the K(d) for the double mutant was not determinable by our technique. Visible-Soret spectra of the carbonyl and cyanyl forms and FT-IR of the carbonyl form of the E8 mutant were similar to those of the wild type; the opposite was true for the GlnE7His and GlnE7His,ProE8Ala mutants, which also differed from wild type in the visible-Soret spectra of their oxidized forms. Models of the effects of the mutations on distal pocket structure were consistent with the experimental findings, particularly the larger effects of the GlnE7His change.     

Activation of p21-activated kinase 6 by MAP kinase kinase 6 and p38 MAP kinase

The p21-activated kinases (PAKs) contain an N-terminal Cdc42/Rac interactive binding domain, which in the group 1 PAKs (PAK1, 2, and 3) regulates the activity of an adjacent conserved autoinhibitory domain. In contrast, the group 2 PAKs (PAK4, 5, and 6) lack this autoinhibitory domain and are not activated by Cdc42/Rac binding, and the mechanisms that regulate their kinase activity have been unclear. This study found that basal PAK6 kinase activity was repressed by a p38 mitogen-activated protein (MAP) kinase antagonist and could be strongly stimulated by constitutively active MAP kinase kinase 6 (MKK6), an upstream activator of p38 MAP kinases. Mutation of a consensus p38 MAP kinase target site at serine 165 decreased PAK6 kinase activity. Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine-proline-tyrosine, TPY) in the activation loop of the PAK6 kinase domain prevented activation by MKK6. PAK6 activation by MKK6 was also blocked by mutation of an autophosphorylated serine (serine 560) in the PAK6 activation loop, indicating that phosphorylation of this site is necessary for MKK6-mediated activation. PAK4 and PAK5 were similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6 suggests that PAK6 plays a role in the cellular response to stress-related signals.     

Activation of wild type p53 function by its mortalin-binding, cytoplasmically localizing carboxyl terminus peptides

The Hsp70 family member mortalin (mot-2/mthsp70/GRP75) binds to a carboxyl terminus region of the tumor suppressor protein p53. By in vivo co-immunoprecipitation of mot-2 with p53 and its deletion mutants, we earlier mapped the mot-2-binding site of p53 to its carboxyl terminus 312-352 amino acid residues. In the present study we attempted to disrupt mot-2-p53 interactions by overexpression of short p53 carboxyl-terminal peptides. We report that p53 carboxyl-terminal peptides (amino acid residues 312-390, 312-352, 323-390, and 323-352) localize in the cytoplasm, whereas 312-322, 337-390, 337-352, and 352-390 locate mostly in the nucleus. Most interestingly, the cytoplasmically localizing p53 peptides harboring the residues 323-337 activated the endogenous p53 function by displacing it from p53-mortalin complexes and relocating it to the nucleus. Such activation of p53 function was sufficient to cause growth arrest of human osteosarcoma and breast carcinoma cells.