Genetic Diversity of <Emphasis Type="Italic">Hevea</Emphasis> IRRDB’81 Collection Assessed by RAPD Markers

The majority of Hevea (Hevea brasiliensis Muell. Arg.) genetic resource in Vietnam derived from the IRRDB’81 germplasm collected in the Amazonian habitats of the genus. Random amplified polymorphic DNA (RAPD) analysis was used to examine the genetic diversity and structure of the IRRDB’81 germplasm. A total of 59 accessions from 13 different districts of the Brazilian states namely Acre, Rondonia, and Mato Grosso were brought into the study using six arbitrarily preselected primers. Sixty-five RAPD band patterns ranging in size from 0.2 to 3.0 kbp were scored for analysis. Differences in the level of DNA polymorphism among the districts and states were revealed. The percentage of the polymorphic DNA fragments calculated for 13 individual districts varied from 15.38 to 70.77%. The mean values of heterozygosity within the district varied from 0.064 to 0.264. Pairwise district Nei’s genetic distance values ranged from 0.046 for Catriquacu and Itanba of Mato Grosso to 0.304 for Tarauaca of Acre and Aracatuba of Mato Grosso. The estimated values of Shannon’s diversity index ranged from 0.093 for the Assis-Brasil district of Acre to 0.389 for the Jiparana district of Rondonia. The analysis of molecular variance (AMOVA) indicated that most of the genetic variations were found among accessions within the districts, while interdistrict variance component accounted for 14.1% only. The low interdistrict differentiation probably implied an extensive gene flow among them. Both the principal coordinate analysis and UPGMA cluster analysis based on genetic distance values revealed a varying degree of separation among the districts and that conformed to geographical origins of Hevea IRRDB’81 collection.     

Functional characterization of the copper-transporting P-type ATPase gene of <Emphasis Type="Italic">Penicillium janthinellum</Emphasis> strain GXCR

Copper (Cu)-transporting P-type ATPase (CTPA) genes have been documented to play an important role in resistance to heavy metals. However, our understanding of roles of CTPA genes of the filamentous fungi was based only on sequence similarity prediction before. In a previous study, we isolated a Penicillum janthinellum strain GXCR of higher tolerance to Cu (200 mM). In this study, we cloned the partial cDNA of CTPA gene, named PcpA, from the strain GXCR. Sequence alignment indicated that the cloned cDNA sequence has the highest identity (94.4%) with a predictive CTPA gene of Aspergillus clavatus. The PcpA-encoded protein, termed PcpA, has classical functional domains of CTPAs, and shows differences from reported CTPAs in some specific sequence motifs and transmembrane regions. Expression of the PcpA was induced by extracellular Cu, cadmium (Cd), and silver (Ag). PcpA RNA interference (RNAi) mutants with a reduced level of PcpA mRNA were more sensitive to Cu, iron, Cd, and Ag than the wild-type (WT) strain GXCR. When grown in the presence of Cu, iron, and Cd, intracellular Cu and iron contents in the PcpA RNAi mutant were significantly (P<0.05) lower than those in the WT; However, intracellular Cd content in the mutant was significantly (P<0.05) higher than that in the WT. Taken together, it can be concluded that the PcpA functions in Cu uptake and homeostasis, iron uptake, and Cd export from the cytosol to the extracytosol.     

Population structure of the Monocelis lineata (Proseriata, Monocelididae) species complex assessed by phylogenetic analysis of the mitochondrial Cytochrome c Oxidase subunit I (COI) gene

Monocelis lineata consists of a complex of sibling species, widespread in the Mediterranean and Atlantic Ocean. Previous genetic analysis placed in evidence at least four sibling species. Nevertheless, this research was not conclusive enough to fully resolve the complex or to infer the phylogeny/phylogeography of the group. We designed specific primers aiming at obtaining partial sequences of the mtDNA gene Cytochrome c Oxidase subunit I (COI) of M. lineata, and have identified 25 different haplotypes in 32 analyzed individuals. The dendrogram generated by Neighbor-Joining analysis confirmed the differentiation between Atlantic and Mediterranean siblings, as well as the occurrence of at least two Mediterranean sibling species. Thus validated, the method here presented appears as a valuable tool in population genetics and biodiversity surveys on the Monocelis lineata complex.     

Production of a reporter transgenic pig for monitoring Cre recombinase activity

The pig is thought to be the most suitable non-human source of organs for xenotransplantation and is widely used as a model of human disease. Using pigs as disease models requires the design of conditional Cre recombinase-loxP gene modifications, which, in turn, requires a Cre-expressing pig with defined patterns of expression controlled by the use of a tissue-specific promoter. In order to monitor Cre recombinant expression in vivo, it is important to create a reporter strain. We have generated reporter a pig that is based on a single vector that drives the ubiquitous expression of the enhanced green fluorescent protein (EGFP). The EGFP gene is expressed only after Cre-mediated excision of loxP-flanked stop sequences. These reporter transgenic pigs will be of great value for monitoring Cre recombinase activity in vivo.     

Mice Lacking the ISG15 E1 Enzyme UbE1L Demonstrate Increased Susceptibility to both Mouse-Adapted and Non-Mouse-Adapted Influenza B Virus Infection

ISG15 functions as a critical antiviral molecule against influenza virus, with infection inducing both the conjugation of ISG15 to target proteins and production of free ISG15. Here, we report that mice lacking the ISG15 E1 enzyme UbE1L fail to form ISG15 conjugates. Both UbE1L^−/− and ISG15^−/− mice display increased susceptibility to influenza B virus infection, including non-mouse-adapted strains. Finally, we demonstrate that ISG15 controls influenza B virus infection through its action within radioresistant stromal cells and not bone marrow-derived cells. Thus, the conjugation of ISG15 to target proteins within stromal cells is critical to its activity against influenza virus.     

Oligomerization of the Macrophage Mannose Receptor Enhances gp120-mediated Binding of HIV-1

C-type lectin receptors expressed on the surface of dendritic cells and macrophages are able to bind glycoproteins of microbial pathogens via mannose, fucose, and N-acetylglucosamine. Langerin on Langerhans cells, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin on dendritic cells, and mannose receptor (MR) on dendritic cells and macrophages bind the human immunodeficiency virus (HIV) envelope protein gp120 principally via high mannose oligosaccharides. These C-type lectin receptors can also oligomerize to facilitate enhanced ligand binding. This study examined the effect of oligomerization of MR on its ability to bind to mannan, monomeric gp120, native trimeric gp140, and HIV type 1 BaL. Mass spectrometry analysis of cross-linked MR showed homodimerization on the surface of primary monocyte-derived dendritic cells and macrophages. Both monomeric and dimeric MR were precipitated by mannan, but only the dimeric form was co-immunoprecipitated by gp120. These results were confirmed independently by flow cytometry analysis of soluble monomeric and trimeric HIV envelope and a cellular HIV virion capture assay. As expected, mannan bound to the carbohydrate recognition domains of MR dimers mostly in a calcium-dependent fashion. Unexpectedly, gp120-mediated binding of HIV to dimers on MR-transfected Rat-6 cells and macrophages was not calcium-dependent, was only partially blocked by mannan, and was also partially inhibited by N-acetylgalactosamine 4-sulfate. Thus gp120-mediated HIV binding occurs via the calcium-dependent, non-calcium-dependent carbohydrate recognition domains and the cysteine-rich domain at the C terminus of MR dimers, presenting a much broader target for potential inhibitors of gp120-MR binding.The mannose receptor (MR)² is a C-type lectin receptor that is expressed on the surface of a variety of cells, including immature monocyte-derived dendritic cells (MDDC), dermal dendritic cells, macrophages, and hepatic endothelial cells. It is a multifunctional protein, involved in antigen recognition and internalization during the early stages of the innate immune response (1) as well as physiological clearance of the endogenous pituitary hormones lutropin and thyrotropin (2, 3). Recognition of foreign antigens occurs via mannose, fucose, and GlcNAc residues (4, 5), which are generally not found as terminal residues on mammalian glycoproteins but are highly abundant on surface proteins of pathogens such as the HIV-1 envelope gp120 (6, 7). Once bound, pathogens can be internalized by endocytosis or phagocytosis, where they are targeted to lysosomes for proteolytic degradation and presentation on major histocompatibility complex class II (8). In immature DCs, soluble recombinant HIV envelope proteins are processed by this pathway, initially binding to both dendritic cell-specific intracellular adhesion molecule 3 grabbing non-integrin (DC-SIGN) and MR and ultimately co-localizing with MR but not DC-SIGN in lysosomes (9). Furthermore, in immature DCs and to a greater extent mature DCs, a proportion of intact HIV-1 enters a unique vesicular compartment that co-localizes with tetraspanin proteins such as CD81 (10, 11). Recently, this compartment has been shown to be continuous with the plasma membrane (11) and does not represent a continuation of the endolysosomal network. Interestingly, this compartment can translocate virus from DCs to CD4 T cells, upon the formation of a virological synapse (10–12). Although viral uptake can occur in DCs independent of HIV env (2), the efficiency of HIV binding and uptake is greatly enhanced by the presence of C-type lectin-env interactions. At least initial binding to DC-SIGN (and most likely also MR) is required for T cell trans-infection (13).Structurally, the extracellular domain of MR consists of an N-terminal cysteine-rich domain (Cys-RD), followed by a fibronectin type II domain and eight carbohydrate recognition domains (CRD) on a single polypeptide backbone (1). Of the eight CRDs, CRD 4–8 have been shown to be required for high affinity binding of ligands containing terminal mannose/fucose/GlcNAc residues, with CRD 4 having demonstrable monosaccharide binding in isolation (14). Binding and release of ligand within the low pH environment of the endolysosomal compartment are also Ca²⁺-dependent. Acid-induced removal of Ca²⁺ binding in CRD 4 and 5 was shown to cause a conformational rearrangement of the domain, resulting in a loss of carbohydrate binding activity (15). In contrast, binding of sulfated carbohydrates to the Cys-RD appears to be Ca²⁺-independent as no Ca²⁺-binding sites were observed in its crystal structure (2, 16).Oligomerization of CLRs such as DC-SIGN (17), Langerin (18), and mannose-binding protein (19) has been reported to be essential for binding of oligosaccharide-bearing ligands. Early studies on MR suggested that it exists solely as a monomeric molecule and that clustering of multiple CRDs within the single polypeptide backbone was necessary for high affinity binding of oligosaccharide moieties (20). However, more recent studies have shown that dimerization is possible in the presence of Ca²⁺ (21) and that an equilibrium may exist between monomeric and dimeric forms on the cell surface (22). It is currently unclear what effect dimerization has on ligand binding to the CRDs; however, there is evidence that dimerization of MR is required for high affinity binding of ligands bearing terminal N-acetylgalactosamine 4-sulfate (GalNAc-4-SO₄) such as lutropin and thyrotropin (22) to the Cys-RD.To date, studies on the oligomerization and ligand binding activity of MR have used solubilized protein from cell lysates (20) or purified recombinant fragments (21). Because the membrane microenvironment can influence protein associations, soluble forms of MR may not necessarily be a true model of the quaternary structure and function of the native protein. Here, we used a well established method of cross-linking (23) on MDDCs, monocyte-derived macrophages (MDMs), and MR-transfected Rat-6 cells to preserve lateral protein-protein interactions between MR on the cell surface prior to solubilization. Mass spectrometry analysis of affinity-purified complexes showed they were homo-oligomers, and further resolution of the complex on a low percentage polyacrylamide gel by SDS-PAGE strongly indicates that they are dimers. Dimerization of MR was also found to be essential for binding mannan, monomeric gp120, native trimeric gp140, and HIV-1 viral particles. Persistence of monomeric gp120 and trimeric gp140 binding to dimeric MR in the presence of EGTA and various CRD and other inhibitors, however, suggested that gp120-mediated HIV-1 binding is not Ca²⁺-dependent and that at least binding probably occurs to both Ca²⁺-dependent and -independent CRDs and also the Cys-RD.     

Cliques in mitotic spindle network bring kinetochore‐associated complexes to form dependence pathway

The mitotic spindle is an essential molecular machine for chromosome segregation during mitosis. Achieving a better understanding of its organization at the topological level remains a daunting task. To determine the functional connections among 137 mitotic spindle proteins, a protein–protein interaction network among queries was constructed. Many hub proteins, which connect more than one query and serve as highly plausible candidates for expanding the mitotic spindle proteome, are ranked by conventional degree centrality and a new subnetwork specificity score. Evaluation of the ranking results by literature reviews and empirical verification of SEPT6, a novel top‐ranked hub, suggests that the subnetwork specificity score could enrich for putative spindle‐related proteins. Topological analysis of this expanded network shows the presence of 30 3‐cliques and six 4‐cliques (fully connected subgraphs) that, respectively, reside in eight kinetochore‐associated complexes, of which seven are evolution conserved. Notably, these complexes strikingly form dependence pathways for the assembly of the kinetochore complex. These analyses indicate the feasibility of using network topology, i.e. cliques, to uncover novel pathways to accelerate our understanding of potential biological processes.     

Two genetically distinct units of Sinomanglietia glauca (Magnoliaceae) detected by chloroplast PCR‐SSCP

Sinomanglietia glauca is a critically endangered species described from Jiangxi Province in the 1990s. Recently two populations were discovered from Yongshun County of west Hunan Province, about 450 km away from those in Jiangxi. Because of the new findings and the poor reproducibility inherent to RAPD and ISSR markers of previous studies, the population structure of this rare species was reanalyzed with chloroplast PCR‐SSCP (single‐stranded conformation polymorphism), including all of four recorded populations. The results showed that two distinct haplotypes characterized Jiangxi and Hunan populations separately, with no genetic variation occurring within regions. We postulated that this surprising pattern might result from habitat fragmentation and demographic bottlenecks during and/or after the Quaternary glaciation. On the basis of the pronounced genetic structure, two evolutionarily significant units (ESUs) were recommended for effective conservation of S. glauca.     

Simvastatin ameliorates established pulmonary hypertension through a heme oxygenase-1 dependent pathway in rats

Background

Simvastatin has been shown to ameliorate pulmonary hypertension by several mechanisms in experimental animal models. In this study, we hypothesized that the major benefits of simvastatin in pulmonary hypertension occur via the heme oxygenase-1 pathway.

Methods

Simvastatin (10 mg/kgw/day) was tested in two rat models of pulmonary hypertension (PH): monocrotaline administration and chronic hypoxia. The hemodynamic changes, right heart hypertrophy, HO-1 protein expression, and heme oxygenase (HO) activity in lungs were measured in both models with and without simvastatin treatment. Tin-protoporphyrin (SnPP, 20 μmol/kg w/day), a potent inhibitor of HO activity, was used to confirm the role of HO-1.

Results

Simvastatin significantly ameliorated pulmonary arterial hypertension from 38.0 ± 2.2 mm Hg to 22.1 ± 1.9 mm Hg in monocrotaline-induced PH (MCT-PH) and from 33.3 ± 0.8 mm Hg to 17.5 ± 2.9 mm Hg in chronic hypoxia-induced PH (CH-PH) rats. The severity of right ventricular hypertrophy was significantly reduced by simvastatin in MCT-PH and CH-PH rats. Co-administration with SnPP abolished the benefits of simvastatin. Simvastatin significantly increased HO-1 protein expression and HO activity in the lungs of rats with PH; however co-administration of SnPP reduced HO-1 activity only. These observations indicate that the simvastatin-induced amelioration of pulmonary hypertension was directly related to the activity of HO-1, rather than its expression.

Conclusion

This study demonstrated that simvastatin treatment ameliorates established pulmonary hypertension primarily through an HO-1-dependent pathway.