Engineering of Stable Recombinant Bacteria for Production of Chiral Medium-Chain-Length Poly-3-Hydroxyalkanoates

In order to scale up medium-chain-length polyhydroxyalkanoate (mcl-PHA) production in recombinant microorganisms, we generated and investigated different recombinant bacteria containing a stable regulated expression system for phaC1, which encodes one of the mcl-PHA polymerases of Pseudomonas oleovorans. We used the mini-Tn5 system as a tool to construct Escherichia coli 193MC1 and P. oleovorans POMC1, which had stable antibiotic resistance and PHA production phenotypes when they were cultured in a bioreactor in the absence of antibiotic selection. The molecular weight and the polydispersity index of the polymer varied, depending on the inducer level. E. coli 193MC1 produced considerably shorter polyesters than P. oleovorans produced; the weight average molecular weight ranged from 67,000 to 70,000, and the polydispersity index was 2.7. Lower amounts of inducer added to the media shifted the molecular weight to a higher value and resulted in a broader molecular mass distribution. In addition, we found that E. coli 193MC1 incorporated exclusively the R configuration of the 3-hydroxyoctanoate monomer into the polymer, which corroborated the enantioselectivity of the PhaC1 polymerase enzyme.     

Normal immune function of monocyte-derived dendritic cells from HIV-infected individuals: implications for immunotherapy.

Dendritic cells (DC) are the most potent cells involved in the generation of primary and secondary immune responses. To assess the feasibility of using autologous DC as immunotherapy for HIV disease, we analyzed a variety of immune parameters using DC isolated from HIV-infected (HIV+) individuals, as well as DC obtained from HIV-uninfected (HIV-) individuals infected in vitro with HIV. After stimulation with recombinant CD40 ligand (CD40LT), cytokine and beta-chemokine production were similar by DC from HIV- donors infected in vitro with the CCR5-using HIV Ba-L strain (n = 8) compared with uninfected DC from the same donors. Production of beta-chemokines, but not of cytokines, was increased by a CXCR4-using IIIB strain-infected DC (n = 7). Stimulation of HIV-infected DC with CD40LT decreased infection in Ba-L-infected DC, but had no effect on IIIB-infected DC. Consistent with this finding, CD40LT down-regulated CCR5 and up-regulated CXCR4 expression on DC. Monocyte-derived DC were also propagated from 15 HIV+ and 13 HIV- donors. They exhibited similar expression of costimulatory molecules and produced similar amounts of IL-12, IL-10, and beta-chemokines, following stimulation. By contrast, stimulated PBMC from HIV+ patients exhibited decreased IL-12 and increased IL-10 production. In summary, phenotype, cytokine secretion, and beta-chemokine production by DC from HIV+ individuals were normal. These cells may prove useful in boosting cellular immune responses in HIV+ individuals.     

A novel gene delivery system targeting cells expressing VEGF receptors

Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors.GV1,GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex.Using pSV2-β-galactosidase as a reporter gene,it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) and human malignant melanoma cell lines (A375) in vitro.In vivo experiments,exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO,human malignant melanoma A375 and human hepatoma graft in nude mice.This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice.These results are correlated with the relevant receptors(flt-1,flk-1/KDR) expression on the targeted cells and tissues.     

Adult Polycystic Kidney Disease in a Rhesus Monkey (Macaca mulatta)

Adult polycystic kidney disease was diagnosed at necropsy in a 16-year-old rhesus monkey dead from renal failure. Both kidneys were enlarged and contained multiple cysts ranging from 0.1 to 4.0 cm in diameter. The age of onset of the clinical signs, terminal uremia, and the gross and histologic findings in this macaque were consistent with adult (type III) polycystic kidney disease of man.     

PTMs in Conversation: Activity and Function of Deubiquitinating Enzymes Regulated via Post-Translational Modifications

Deubiquitinating enzymes (DUBs) constitute a diverse protein family and their impact on numerous biological and pathological processes has now been widely appreciated. Many DUB functions have to be tightly controlled within the cell, and this can be achieved in several ways, such as substrate-induced conformational changes, binding to adaptor proteins, proteolytic cleavage, and post-translational modifications (PTMs). This review is focused on the role of PTMs including monoubiquitination, sumoylation, acetylation, and phosphorylation as characterized and putative regulative factors of DUB function. Although this aspect of DUB functionality has not been yet thoroughly studied, PTMs represent a versatile and reversible method of controlling the role of DUBs in biological processes. In several cases PTMs might constitute a feedback mechanism insuring proper functioning of the ubiquitin proteasome system and other DUB-related pathways.     

Properties of Engineered Poly-3-Hydroxyalkanoates Produced in Recombinant Escherichia coli Strains

To prepare medium-chain-length poly-3-hydroxyalkanoates (PHAs) with altered physical properties, we generated recombinant Escherichia coli strains that synthesized PHAs with altered monomer compositions. Experiments with different substrates (fatty acids with different chain lengths) or different E. coli hosts failed to produce PHAs with altered physical properties. Therefore, we engineered a new potential PHA synthetic pathway, in which ketoacyl-coenzyme A (CoA) intermediates derived from the β-oxidation cycle are accumulated and led to the PHA polymerase precursor R-3-hydroxyalkanoates in E. coli hosts. By introducing the poly-3-hydroxybutyrate acetoacetyl-CoA reductase (PhbB) from Ralstonia eutropha and blocking the ketoacyl-CoA degradation step of the β-oxidation, the ketoacyl-CoA intermediate was accumulated and reduced to the PHA precursor. Introduction of the phbB gene not only caused significant changes in the monomer composition but also caused changes of the physical properties of the PHA, such as increase of polymer size and loss of the melting point. The present study demonstrates that pathway engineering can be a useful approach for producing PHAs with engineered physical properties.     

Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles

Chloroplasts contain lipoprotein particles termed plastoglobules. Plastoglobules are generally believed to have little function beyond lipid storage. Here we report on the identification of plastoglobule proteins using mass spectrometry methods in Arabidopsis thaliana. We demonstrate specific plastoglobule association of members of the plastid lipid-associated proteins/fibrillin family as well as known metabolic enzymes, including the tocopherol cyclase (VTE1), a key enzyme of tocopherol (vitamin E) synthesis. Moreover, comparative analysis of chloroplast membrane fractions shows that plastoglobules are a site of vitamin E accumulation in chloroplasts. Thus, in addition to their lipid storage function, we propose that plastoglobules are metabolically active, taking part in tocopherol synthesis and likely other pathways.     

河西走廊春季不同盐碱土壤中微生物数量、酶活性与理化因子的关系

[目的]揭示盐碱土壤微生物量与土壤因子间的关系.[方法]选择河西走廊不同盐碱程度的11个样点在春季进行采样,研究了土壤的微生物数量、酶活和理化性质,并对其进行方差分析、简单相关分析、逐步回归分析和主成分分析.[结果]河西地区原生盐碱地、次生盐碱地与农田土在土壤理化性质和土壤微生物数量等方面均有差异;河西地区土壤较贫瘠,土壤微生物数量较低,且分布有规律性,即原生盐碱土＜次生盐碱土＜农田土;放线菌、真菌、碱性磷酸酶、脲酶和有效磷5个因子是引起土壤微生物数量、酶活性与理化因子之间相关性的主要因素.[结论]结果证实河西地区盐碱土壤中磷的循环很大程度上影响着土壤微生物数量.     

Correction to: Reef-associated fishes have more maneuverable body shapes at a macroevolutionary scale

Coral Reefs - This erratum has been initiated as several formatting errors were overlooked during proofing stage by vendor.     

Influence of niche characteristics and forest type on fern species richness,abundance and plant size along an elevational gradient in Costa Rica

An analysis of the fern vegetation on 156 plots along an elevational gradient (45-3400 m) in undisturbed forests in Costa Rica, Central America, showed a hump-shaped pattern of species richness with a maximum of up to 68 species per 400 m2 at mid-elevations. This study documents the contribution of specific habitats (forest types: ridges, ravines) and niches within them (dead wood, rocks, growth zones in trees) to the local fern richness and the relation of species richness to elevation and climatic variables. Forests along ravines showed significantly higher species richness, presumably caused by high environmental humidity. The mean number of individuals of occupied niches per species increased significantly with elevation, suggesting that the niche breadth of species increased and that the differentiation of niches decreased with elevation. Both findings may explain the reduced fern species richness towards and above the upper treeline, but not at low elevations. The key factors for the decreases of species richness at the extremes of the gradient are likely to involve climatic conditions.