A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees

Three percent of the world's population is chronically infected with the hepatitis C virus (HCV) and at risk of developing liver cancer. Effective cellular immune responses are deemed essential for spontaneous resolution of acute hepatitis C and long-term protection. Here we describe a new T-cell HCV genetic vaccine capable of protecting chimpanzees from acute hepatitis induced by challenge with heterologous virus. Suppression of acute viremia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8(+) T lymphocytes that cross-reacted with vaccine and virus epitopes. These findings show that it is possible to elicit effective immunity against heterologous HCV strains by stimulating only the cellular arm of the immune system, and suggest a path for new immunotherapy against highly variable human pathogens like HCV, HIV or malaria, which can evade humoral responses.     

Inhibitory effect of obesity on gonadotropin, estradiol, and inhibin B levels in fertile women

Objective: To examine whether obesity and insulin resistance have an independent effect on the gonadotropin, estradiol, and inhibin B serum levels and follicle count in the early follicular phase of fertile women with a wide range of BMI and without signs of hyperandrogenism. Research Methods and Procedures: Twenty‐two overweight and obese (BMI ≥25.0 kg/m²) women and 10 normal‐weight (BMI <25.0 kg/m²) women, all having apparently normal fertility, were studied. Serum concentrations of follicle‐stimulating hormone (FSH), luteinizing hormone (LH), estradiol, inhibin B, and insulin, level of insulin resistance (estimated by homeostasis model assessment for insulin resistance), and follicle count were measured during the early follicular phase (Days 2 to 5 of the menstrual cycle). Results: Overweight women showed lower FSH (p < 0.001), LH (p < 0.001), and inhibin B (p < 0.05) levels compared with normal‐weight women, whereas estradiol concentrations and follicle count were not significantly different between the two groups. When normal‐weight and overweight women were examined as a group and multiple regression analyses were performed, estradiol showed a negative association with BMI (or waist circumference) (p < 0.05) and a positive correlation with LH (p < 0.05) and FSH (p < 0.05); inhibin B maintained a positive association only with estradiol (p < 0.05); and FSH and LH showed a negative correlation with BMI (or waist circumference) (p < 0.001 and p < 0.01, respectively). Discussion: Overweight and obese fertile women have lower FSH, LH, inhibin B, and estradiol levels in the early follicular phase, with a possible direct inhibitory effect of body mass on gonadotropin and estradiol production, independently of age, insulin (concentrations and sensitivity), and other hormones. By contrast, the number of ovary follicles does not seem to be influenced by insulin and body mass in these patients.     

Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes

Mammals that degrade uric acid are not affected by gout or urate kidney stones. It is not fully understood how they convert uric acid into the much more soluble allantoin. Until recently, it had long been thought that urate oxidase was the only enzyme responsible for this conversion. However, detailed studies of the mechanism and regiochemistry of urate oxidation have called this assumption into question, suggesting the existence of other distinct enzymatic activities. Through phylogenetic genome comparison, we identify here two genes that share with urate oxidase a common history of loss or gain events. We show that the two proteins encoded by mouse genes catalyze two consecutive steps following urate oxidation to 5-hydroxyisourate (HIU): hydrolysis of HIU to give 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and decarboxylation of OHCU to give S-(+)-allantoin. Urate oxidation produces racemic allantoin on a time scale of hours, whereas the full enzymatic complement produces dextrorotatory allantoin on a time scale of seconds. The use of these enzymes in association with urate oxidase could improve the therapy of hyperuricemia.     

Structure-activity relationships of 1,5-biaryl pyrroles as EP1 receptor antagonists

The preliminary SAR of a series of novel 1,5-biaryl pyrrole EP1 receptor antagonists derived from compound 1 is described. Replacement of the benzyl group of 1 with isosteric groups was investigated. The most effective replacement was found to be the isobutyl group. The cyclopentylmethyl and cyclohexylmethyl groups were also effective benzyl replacements. The cyclohexylmethyl derivative 19 demonstrated the lowest metabolic clearance within this series. In addition, several high affinity substituted benzyl analogues were also identified. Compound 39 was found to have good bioavailability in rats and demonstrated efficacy in the established FCA preclinical model of inflammatory pain with a calculated ED50 of 9.2mg/kg.     

alpha-Tocopherol affects neuronal plasticity in adult rat dentate gyrus: the possible role of PKCdelta

Hippocampus dentate gyrus (DG) is characterized by neuronal plasticity processes in adulthood, and polysialylation of NCAM promotes neuronal plasticity. In previous investigations we found that alpha-tocopherol increased the PSA-NCAM-positive granule cell number in adult rat DG, suggesting that alpha-tocopherol may enhance neuronal plasticity. To verify this hypothesis, in the present study, structural remodeling in adult rat DG was investigated under alpha-tocopherol supplementation conditions. PSA-NCAM expression was evaluated by Western blotting, evaluation of PSA-NCAM-positive granule cell density, and morphometric analysis of PSA-NCAM-positive processes. In addition, the optical density of synaptophysin immunoreactivity and the synaptic profile density, examined by electron microscopy, were evaluated. Moreover, considering that PSA-NCAM expression has been found to be related to PKCdelta activity and alpha-tocopherol has been shown to inhibit PKC activity in vitro, Western blotting and immunohistochemistry followed by densitometry were used to analyze PKC. Our results demonstrated that an increase in PSA-NCAM expression and optical density of DG molecular layer synaptophysin immunoreactivity occurred in alpha-tocopherol-treated rats. Electron microscopy analysis showed that the increase in synaptophysin expression was related to an increase in synaptic profile density. In addition, Western blotting revealed a decrease in phospho-PKC Pan and phospho-PKCdelta, demonstrating that alpha-tocopherol is also able to inhibit PKC activity in vivo. Likewise, immunoreactivity for the active form of PKCdelta was lower in alpha-tocopherol-treated rats than in controls, while no changes were found in PKCdelta expression. These results demonstrate that alpha-tocopherol is an exogenous factor affecting neuronal plasticity in adult rat DG, possibly through PKCdelta inhibition.     

A strained DNA binding helix is conserved for site recognition,folding nucleation,and conformational modulation

Nucleic acid recognition is often mediated by α‐helices or disordered regions that fold into α‐helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical α‐helix is stabilized at the N‐terminus, while the PII forms at the C‐terminus half of the peptide. Re‐examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N‐terminus half is a canonical α‐helix, while the C‐terminal half adopts a 3₁₀ helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 10⁴‐fold lower affinity, and 500‐fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained α‐helix conformation, “presented” by this unique architecture. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 432–443, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Mapping Loci Associated With Tail Color and Sex Determination in the Short-Lived Fish Nothobranchius furzeri

The African fish Nothobranchius furzeri is the shortest-lived vertebrate species that can reproduce in captivity, with a median life span of 9–11 weeks for the shortest-lived strain. Natural populations of N. furzeri display differences in life span, aging biomarkers, behavior, and color, which make N. furzeri a unique vertebrate system for studying the genetic basis of these traits. We mapped regions of the genome involved in sex determination and tail color by genotyping microsatellite markers in the F₂ progeny of a cross between a short-lived, yellow-tailed strain and a long-lived, red-tailed strain of N. furzeri. We identified one region linked with the yellow/red tail color that maps close to melanocortin 1 receptor (mc1r), a gene involved in pigmentation in several vertebrate species. Analysis of the segregation of sex-linked markers revealed that N. furzeri has a genetic sex determination system with males as the heterogametic sex and markedly reduced recombination in the male sex-determining region. Our results demonstrate that both naturally-evolved pigmentation differences and sex determination in N. furzeri are controlled by simple genetic mechanisms and set the stage for the molecular genetic dissection of factors underlying such traits. The microsatellite-based linkage map we developed for N. furzeri will also facilitate analysis of the genetic architecture of traits that characterize this group of vertebrates, including short life span and adaptation to extreme environmental conditions.THE Nothobranchius fish species are present in eastern and southeastern Africa, where they populate ephemeral water pools that often undergo complete desiccation during the dry season (Terzibasi et al. 2008; Reichard et al. 2009; Wildekamp 2009). Nothobranchius species tend to live in extreme habitats and have evolved unique adaptations to harsh environmental conditions, including extremely short life cycles, resistance to a wide range of temperatures and water salinity, embryonic development that does not require the presence of water, and a developmental diapause that allows embryos to survive for months in dry conditions (Wourms 1972; Inglima et al. 1981; Genade et al. 2005).Nothobranchius furzeri is the shortest-lived species of the Nothobranchius genus, with an intergeneration time of 40 days, a median life span of 9–11 weeks, and a maximum life span of 12–15 weeks for the shortest-lived strain GRZ (Valdesalici and Cellerino 2003; Genade et al. 2005; Valenzano et al. 2006; Terzibasi et al. 2008, 2009; Hartmann et al. 2009). Natural populations of N. furzeri can vary substantially in phenotypic traits. For example, N. furzeri strains derived from Zimbabwe and northern Mozambique (e.g., GRZ) exhibit a shorter life span than strains derived from more humid areas in southern Mozambique (e.g., MZM-0403) under controlled conditions (Terzibasi et al. 2008). The extremely short life cycle of N. furzeri and the presence of natural populations with phenotypic variations make this species a promising model system for studying aging and adult-specific traits, including color and behavior.The color pattern of the adult male tail differs among N. furzeri strains. GRZ males show a yellow submarginal band and a black marginal band (yellow morph) whereas MZM-0403 males display a broad red band (red morph) in the caudal fin (Figure 1A). This dichromatism is present in natural populations of N. furzeri (Terzibasi et al. 2008; Reichard et al. 2009). Similar color polymorphism among males is also observed in other species of Nothobranchius (Wildekamp 2009) and in other fish species, including guppies and cichlids (Hughes et al. 1999; Brooks and Endler 2001; Maan et al. 2004). Differences in male color morphs within the same species are associated with sexual preference by females, different recognition by predators depending on the habitat, and differential susceptibility to pathogens (Price et al. 2008), which could all influence the evolution of this trait. Despite the widespread variation in Nothobranchius coloration, the genetic basis of this trait is unknown.Open in a separate windowFigure 1.—Cross between two strains of N. furzeri that differ in color and life span (A) Color phenotypes of GRZ and MZM-0403. (B) A yellow-tailed, short-lived male GRZ and a red-tailed, long-lived female MZM-0403 were the founders of cross 1.Genetic information on N. furzeri is still limited. The N. furzeri genome is 1.6–1.9 Gb in size and is characterized by a high repeat content (45%) (Reichwald et al. 2009). N. furzeri has 19 chromosomes, but no morphologically discernible sex chromosomes (Reichwald et al. 2009). The sex determination system in N. furzeri has not been characterized yet. Sex can be determined either genetically or environmentally in fish (Volff 2005; Marshall Graves 2008). For example, medaka, platyfish, guppy, and sticklebacks all have recently evolved genetic sex-determining systems (Volff and Schartl 2002; Peichel et al. 2004; Shapiro et al. 2009; Tripathi et al. 2009a), whereas zebrafish do not have a clear genetic basis of sex determination (von Hofsten and Olsson 2005).Genetic studies in N. furzeri would greatly benefit from the building of a linkage map in this species of fish. However, to date there is no linkage map available for N. furzeri or for any Nothobranchius species, although linkage maps have been generated for fish of the same order, e.g., Poecilia reticulata (guppy) and Xiphophorus maculatus (platyfish) (Khoo et al. 2003; Walter et al. 2004; Tripathi et al. 2009b), and of the same superorder, e.g., Oryzias latipes (medaka) (Wada et al. 1995).Here, we report a microsatellite-based linkage map for N. furzeri using a genetic cross between the short-lived yellow-tailed GRZ strain and the long-lived red-tailed MZM-0403 strain. This N. furzeri linkage map allowed us to map the male-specific tail color trait on linkage group (LG) V. Synteny analysis revealed that LG V has homology to a region of the medaka genome that contains the melanocortin 1 receptor (mc1r) gene, which is known to play a key role in vertebrate pigmentation. We identified a sequence polymorphism in mc1r between the two strains of N. furzeri, allowing us to map mc1r on LG V. This analysis revealed that mc1r is located in close proximity to the color locus, but that the sequence polymorphism is probably not causative for the color difference. We also found that sex is genetically determined in N. furzeri, with males as the heterogametic sex. The sex-determining region is located on LG XIII and is characterized by male-specific suppression of recombination. Our findings will be pivotal for the identification of the genetic determinants of color in N. furzeri and for expanding our knowledge about sex-determination mechanisms in vertebrates. Due to the array of intraspecific phenotypic differences displayed by the various populations of N. furzeri, this linkage map will also be a key tool for mapping phenotypic variation in this short-lived vertebrate species, including differences in life span.     

Human cytomegalovirus productively infects adrenocortical cells and induces an early cortisol response

Following our recent findings on the presence of human cytomegalovirus (HCMV) in the normal human adrenal cortex and in adrenocortical tumors, especially in cortisol‐secreting tumors, aim of the present study was to investigate the direct effects of HCMV infection on human adrenocortical cells. To this aim, both clinical isolates and laboratory strains of HCMV were used to assess the early effects of infection on human adrenocortical cell morphology, proliferation, gene expression, and steroidogenic function. Both clinical and laboratory HCMV strains could infect and replicate in primary human adrenocortical cell cultures and in adrenocortical carcinoma cell lines, leading to cytopathic changes. Most importantly, in the first hours post‐infection (p.i.), adrenocortical cells showed a significant increase of cortisol and estrogen production, paralleled by up‐regulation of steroidogenic acute regulatory protein and expression of steroidogenic enzymes involved in the last steps of adrenal steroidogenesis. This effect was probably due to HCMV immediate‐early gene expression, since it was most evident in the early phases p.i. and UV‐inactivated viral particles did not affect hormone production. Moreover, the effect on steroidogenesis was HCMV specific, since it was not observed after infection with herpes simplex virus. These data suggest that human adrenocortical cells are permissive to HCMV infection and acutely respond to infection with increased cortisol production. An acute glucocorticoid response is typically triggered by infections and is considered to be critical to host defense against pathogens, although, in the case of HCMV infection, it might also enhance viral replication and reactivation from latency. J. Cell. Physiol. 221: 629–641, 2009. © 2009 Wiley‐Liss, Inc.     

Helicospiral growth in the whip black coral Cirrhipathes sp. (Antipatharia, Antipathidae)

The coiling pattern of the whip black coral Cirrhipathes sp. (Antipatharia, Antipathidae) was studied in four sites of the Bunaken Marine Park (North Sulawesi, Indonesia) characterized by different hydrodynamic conditions. The colonies show a helicospiral growth, with the polyps irregularly arranged on the external convex side of the turns of the stem. Only the colonies living in a very slow current environment reach the greatest lengths (up to 5 m) and show the highest number of rotations (up to 10, 3600°). The turns shape changes along the stem of the colonies, from the base to the apex, as evidenced by the ratio between the pitch and the diameter of each coil. In particular, the turns close to the basal plate have a smaller diameter and a larger pitch, while in the most distal turns, the diameter increases and the pitch decreases concurrently. We hypothesize that the shape variation of the whorls is triggered by the relationship between the growing stem and the intensity of the prevailing current. In this way, the colony can initially grow fast, moving away from the bottom, and then extend into the current, maximizing the plankton capture.