Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.     

Isolation and Characterization of Polymeric Galloyl-Ester-Degrading Bacteria from a Tannery Discharge Place

The culturable bacteria colonizing the rhizosphere of plants growing in the area of discharge of a tannery effluent were characterized. Relative proportions of aerobic, denitrifying, and sulfate-reducing bacteria were determined in the rhizosphere of Typha latifolia, Canna indica, and Phragmites australis. Aerobic bacteria were observed to be the most abundant group in the rhizosphere, and plant type did not seem to influence the abundance of the bacterial types analyzed. To isolate bacteria able to degrade polyphenols used in the tannery industry, enrichments were conducted under different conditions. Bacterial cultures were enriched with individual polyphenols (tannins Tara, Quebracho, or Mimosa) or with an undefined mixture of tannins present in the tannery effluent as carbon source. Cultures enriched with the effluent or Tara tannin were able to degrade tannic acid. Six bacterial isolates purified from these mixed cultures were able to use tannic acid as a sole carbon source in axenic culture. On the basis of 16S ribosomal DNA sequence analysis, these isolates were closely related to organisms belonging to the taxa Serratia, Stenotrophomonas maltophilia, Klebsiella oxytoca, Herbaspirillum chlorophenolicum, and Pseudomonas putida.     

High-resolution molecular epidemiology and evolutionary history of HIV-1 subtypes in Albania

Background

HIV-1 epidemic in Western Europe is largely due to subtype B. Little is known about the HIV-1 in Eastern Europe, but a few studies have shown that non-B subtypes are quite common. In Albania, where a recent study estimated a ten-fold increase of AIDS incidence during the last six years, subtype A and B account for 90% of the know infections.

Methodology/Principal Findings

We investigated the demographic history of HIV-1 subtype A and B in Albania by using a statistical framework based on coalescent theory and phylogeography. High-resolution phylogenetic and molecular clock analysis showed a limited introduction to the Balkan country of subtype A during the late 1980s followed by an epidemic outburst in the early 1990s. In contrast, subtype B was apparently introduced multiple times between the mid-1970s and mid-1980s. Both subtypes are growing exponentially, although the HIV-1A epidemic displays a faster growth rate, and a significantly higher basic reproductive number R₀. HIV-1A gene flow occurs primarily from the capital Tirane, in the center of the country, to the periphery, while HIV-1B flow is characterized by a balanced exchange between center and periphery. Finally, we calculated that the actual number of infections in Albania is at least two orders of magnitude higher than previously thought.

Conclusions/Significance

Our analysis demonstrates the power of recently developed computational tools to investigate molecular epidemiology of pathogens, and emphasize the complex factors involved in the establishment of HIV-1 epidemics. We suggest that a significant correlation exists between HIV-1 exponential spread and the socio-political changes occurred during the Balkan wars. The fast growth of a relatively new non-B epidemic in the Balkans may have significant consequences for the evolution of HIV-1 epidemiology in neighboring countries in Eastern and Western Europe.     

Linkage and Association Mapping for Two Major Traits Used in the Maritime Pine Breeding Program: Height Growth and Stem Straightness

BackgroundIncreasing our understanding of the genetic architecture of complex traits, through analyses of genotype-phenotype associations and of the genes/polymorphisms accounting for trait variation, is crucial, to improve the integration of molecular markers into forest tree breeding. In this study, two full-sib families and one breeding population of maritime pine were used to identify quantitative trait loci (QTLs) for height growth and stem straightness, through linkage analysis (LA) and linkage disequilibrium (LD) mapping approaches.ResultsThe populations used for LA consisted of two unrelated three-generation full-sib families (n = 197 and n = 477). These populations were assessed for height growth or stem straightness and genotyped for 248 and 217 markers, respectively. The population used for LD mapping consisted of 661 founders of the first and second generations of the breeding program. This population was phenotyped for the same traits and genotyped for 2,498 single-nucleotide polymorphism (SNP) markers corresponding to 1,652 gene loci. The gene-based reference genetic map of maritime pine was used to localize and compare the QTLs detected by the two approaches, for both traits. LA identified three QTLs for stem straightness and two QTLs for height growth. The LD study yielded seven significant associations (P ≤ 0.001): four for stem straightness and three for height growth. No colocalisation was found between QTLs identified by LA and SNPs detected by LD mapping for the same trait.ConclusionsThis study provides the first comparison of LA and LD mapping approaches in maritime pine, highlighting the complementary nature of these two approaches for deciphering the genetic architecture of two mandatory traits of the breeding program.     

Cloning of histidine genes of Azospirillum brasilense: organization of the ABFH gene cluster and nucleotide sequence of the hisB gene

Summary A cluster of four Azospirillum brasilense histidine biosynthetic genes, hisA, hisB, hisF and hisH, was identified on a 4.5 kb DNA fragment and its organization studied by complementation analysis of Escherichia coli mutations and nucleotide sequence. The nucleotide sequence of a 1.3 kb fragment that complemented the E. coli hisB mutation was determined and an ORF of 624 nucleotides which can code for a protein of 207 amino acids was identified. A significant base sequence homology with the carboxyterminal moiety of the E. coli hisB gene (0.53) and the Saccharomyces cerevisiae HIS3 gene (0.44), coding for an imidazole glycerolphosphate dehydratase activity was found. The amino acid sequence and composition, the hydropathic profile and the predicted secondary structures of the yeast, E. coli and A. brasilense proteins were compared. The significance of the data presented is discussed.Abbreviations IGP imidazole glycerolphosphate - HP histidinolphosphate     

Soil–plant–atmosphere conditions regulating convective cloud formation above southeastern US pine plantations

Loblolly pine trees (Pinus taeda L.) occupy more than 20% of the forested area in the southern United States, represent more than 50% of the standing pine volume in this region, and remove from the atmosphere about 500 g C m per year through net ecosystem exchange. Hence, their significance as a major regional carbon sink can hardly be disputed. What is disputed is whether the proliferation of young plantations replacing old forest in the southern United States will alter key aspects of the hydrologic cycle, including convective rainfall, which is the focus of the present work. Ecosystem fluxes of sensible () and latent heat (LE) and large‐scale, slowly evolving free atmospheric temperature and water vapor content are known to be first‐order controls on the formation of convective clouds in the atmospheric boundary layer. These controlling processes are here described by a zero‐order analytical model aimed at assessing how plantations of different ages may regulate the persistence and transition of the atmospheric system between cloudy and cloudless conditions. Using the analytical model together with field observations, the roles of ecosystem and LE on convective cloud formation are explored relative to the entrainment of heat and moisture from the free atmosphere. Our results demonstrate that cloudy–cloudless regimes at the land surface are regulated by a nonlinear relation between the Bowen ratio and root‐zone soil water content, suggesting that young/mature pines ecosystems have the ability to recirculate available water (through rainfall predisposition mechanisms). Such nonlinearity was not detected in a much older pine stand, suggesting a higher tolerance to drought but a limited control on boundary layer dynamics. These results enable the generation of hypotheses about the impacts on convective cloud formation driven by afforestation/deforestation and groundwater depletion projected to increase following increased human population in the southeastern United States.     

南极Gerlache海峡海鸟的取食集群及食性

1997年12月至1998年2月,我们对南极半岛席尔瓦角善于飞翔海鸟与企鹅的取食关联性进行了研究,同时调查了取食集团中主要鸟种的食性.发现每个取食集团中有35.6-37.0只善于飞翔的海鸟,其中几乎都有纹颊企鹅群 (Pygoscelis antarctica),黑背鸥(Larus dominicanus)、灰贼鸥(Catharacta maccormicki)、花斑鹱(Daption capensis)和巨鹱(Macronectes giganteus)是各集团中最常见的鸟类.各取样单元内有相关性的种数随季节变化而减少,一些种类的减少与特定的物候期有关.南极磷虾(Euphausia superba)是绝大部分飞翔海鸟的主要食物,研究发现黑背鸥与纹颊企鹅所捕食的南极磷虾的大小最为接近.飞翔海鸟的觅食行为表明:在海面上短时停留的飞翔海鸟也能够成功捕捉到磷虾,这可能与磷虾躲避企鹅的捕食有关[动物学报 53(3):425-430,2007].     

Angiogenesis and diabetes: different responses to pro-angiogenic factors in the chorioallantoic membrane assay

Hyperglycemia induces defects in angiogenesis without alteration in the expression of major vascular growth factors in the chicken chorioallantoic membrane (CAM) model. A direct negative effect of hyperglycemia on angiogenesis may participate in failures of "therapeutic angiogenesis" trials. Here, we tested the hypothesis that the response to pro-angiogenic molecules such as angiotensin-converting enzyme (ACE), endothelin-1 (ET-1), and vascular endothelial growth factor-A (VEGF) is altered by hyperglycemia. Transfected (Chinese hamster ovary [CHO] or human embryonic kidney [HEK]) cells overexpressing ACE, ET-1, or VEGF were deposed onto the CAM of hyperglycemic or control embryos. The proangiogenic effect was evaluated 3 d later by angiography and histological analyses. Gene expression in response to these factors was assessed by in situ hybridization. Only VEGF overexpression evoked a proangiogenic response in the CAM from hyperglycemic embryos, upregulating the expression of endogenous VEGF, VEGF-R2, and Tie-2, all of them related to activation of endothelial cells. In conclusion, in a model where hyperglycemia does not alter the major vascular growth factor expression, the negative effect of diabetes on capillary density was overcome only by VEGF overexpression, whereas responses to other vasoactive peptides were practically abolished under hyperglycemic conditions.     

Human conjunctival epithelial precursor cells and their progeny in 3D organotypic culture

We report on an in vitro organ culture method to investigate human conjunctival epithelial basal precursor cells and their progeny within a more natural three-dimensional microenvironment. Conjunctival fragments were cultured on gelatin sponges in medium with 10% FBS. The conjunctival phenotype of the epithelium was confirmed by the expression and distribution of a panel of markers (p63, CK-13/CK-10, CK-19, Ki-67, PAS for goblet cells, CD45 for infiltrating interlamellar leukocytes and nestin for mesenchymal and ocular epithelial precursor cells). After 7 days, the epithelium had exfoliated its superficial layers (mostly CK-19( )positive cells and all goblets), maintaining only 1-2 layers of basal/parabasal cells, p63, CK-13/CK-10 and nestin positive cells, firmly attached to the specimen. After 14 days, a new multilayered epithelium was formed, consisting of p63, CK-13/CK-10, nestin positive cells and in the high-zone CK-19 positive cells with new goblets. Additionally, we found interlamellar leukocytes which had probably migrated from capillaries that continued to be well maintained in the subepithelial stroma. Cells dispersed from conjunctival epithelium and co-cultured with feeder post-mitotic NIH3T3 fibroblasts formed mosaics displaying a basal epithelial phenotype. These cells expressed CD133 as revealed by RT-PCR. These organ cultures provide new opportunities to investigate epithelial reconstitution of the conjunctival surface and changes that may have occurred to their stem/precursor cells during adaptation to varying conditions in vitro.