家蚕色氨酸羟化酶(TRH)基因的克隆及表达特性分析（英文）

5-羟色胺(5-hydroxytryptamine, 5-HT)是生物界广泛分布的信号分子,涉及动物的重要行为。5-HT是色氨酸羟化酶(Tryptophan hydroxylase, TRH)将L-色氨酸羟化为5-羟-L-色氨酸,5-羟-L-色氨酸随即被多巴脱羧酶(Aromatic L-amino acid decarboxylase, DDC)脱羧而成。TRH作为5-HT合成的限速酶,在无脊椎动物神经调控中具有重要地位。鳞翅目昆虫中TRH的功能研究并不多。在家蚕中克隆了家蚕TRH (Bombyx mori TRH, BmTRH)的cDNA序列1667bp,其中包含1632bp的开放读码框(Openreadingframe,ORF)。人类TPH或者果蝇TRH(Drosophila TRH, DmTRH)与BmTRH有高度相似性,尤其BmTRH和DmTRH之间大多数氨基酸保守说明它们在系统发育上的密切关系并可能有相似功能。基因表达分析显示BmTRH主要表达于头部和中枢神经组织,免疫组织化学和Western blotting结果显示BmTRH只存在于神经组织中,即BmTRH可能仅参与家蚕的神经活动。此外,家蚕DDC(B.moridecarboxylase,BmDDC)和蛋白具有TRH活性的苯丙氨酸羟化酶基因(Phenylalanine hydroxylase, BmPAH)也在中枢神经系统中有表达,暗示家蚕神经系统5-HT的合成与果蝇中不同,可能有两种不同的调控机制。     

PTP1B inhibitor improves both insulin resistance and lipid abnormalities in vivo and in vitro

PTP1B is a negative regulator of insulin signaling pathway. This study investigated the effects of compound CCF06240, a PTP1B inhibitor, on insulin sensitivity and lipid metabolic abnormalities in vivo and in vitro, respectively. The insulin resistant IRM mouse model was induced by HFD. The responses to insulin were determined by OGTT, ITT, and hyperinsulinemic-euglycemic clamp test. The body weight and the levels of serum TC and TG were measured to estimate the lipid metabolism in vivo. Recombinant human GST-PTP1B protein was used to measure the inhibition of CCF06240 on PTP1B activity. The hepatocyte lipid accumulation was induced by high concentrations of FFA and insulin in HepG(2) cells, and evaluated by the Oil Red O method. In IRM mice, the insulin resistance was improved; the body weight and the levels of TC and TG were also reduced by oral CCF06240 administration. In lipid accumulated model cells, CCF06240 was found to reverse the increased PTP1B activity, enhance the insulin-induced tyrosine phosphorylation in insulin signaling pathway, attenuate the FFA-insulin-induced cellular lipid accumulation, and down-regulate the expressions of genes related fatty acid synthesis. These results demonstrated that the PTP1B inhibitor, compound CCF06240, could increase insulin sensitivity through the regulation of insulin signaling pathway, and decrease FFA-insulin-induced hepatocytes lipid accumulation by reducing fatty acid syntheses.     

基于高通量测序分析盐角草根部内生细菌多样性及动态规律

【目的】探讨盐角草根部内生细菌群落多样性特征,揭示内生细菌群落结构在宿主关键发育期动态变化规律。【方法】通过罗氏454高通量测序获得内生细菌16S r RNA片段,然后进行生物信息分析。【结果】共获得20363条16S r RNA基因序列。各样品中可操作分类单元(operational taxonomic units,OTUs)在552–941之间。根部内生细菌群落主要包括4个门,其中Proteobacteri门占主导地位,其余依次是Firmicutes,Actinobacteria,Bacteroidetes。在Proteobacteria门中,Gammaproteobacteria是第一大纲,其后是Betaproteobacteria纲。宿主5个发育时期共同拥有7个细菌属,包括Azomonas,Serratia,Pantoea,Serpens,Pseudomonas,Halomonas,Kushneria。整体上看,Gammaproteobacteria纲在宿主5个发育时期呈现增长趋势。优势菌属在5个发育期存在差异,分别为Delftia,Kushneria,Serratia,Pantoea,Erwinia。所有文库总共含2108个特异OTUs,共同拥有5个相同OTUs。花期OTUs数量最多,结种期内生细菌多样性降低。在宿主的5个发育时期中,土壤p H、月均温和土壤盐含量这3个环境因子组成的集合对其内生细菌群落变化具有显著影响。【结论】盐角草内生细菌群落多样性丰富,宿主发育期决定了内生细菌群落结构。     

HIF-1α acts downstream of TNF-α to inhibit vasodilator-stimulated phosphoprotein expression and modulates the adhesion and proliferation of breast cancer cells

Metastasis is the leading cause of death in breast cancer patients. Recent evidence suggests that inflammation-related cytokine tumor necrosis factor-alpha (TNF-α) is implicated in tumor invasion and metastasis, but the mechanism of its involvement remains elusive. In this study, we employed MCF-7 breast cancer cells as an experimental model to demonstrate that TNF-α inhibits breast cancer cell adhesion and cell proliferation through hypoxia inducible factor-1alpha (HIF-1α) mediated suppression of vasodilator-stimulated phosphoprotein (VASP). We observed that TNF-α treatment attenuated the adhesion and proliferation of MCF-7 cells it also dramatically increased HIF-1α expression and decreased VASP expression. Through a variety of approaches, including promoter assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP), we identified VASP as a direct target gene of HIF-1α. In addition, we confirmed that HIF-1α mediated the repression of VASP expression by TNF-α in MCF-7 cells. We also demonstrated that exogenous VASP expression or knockdown of HIF-1α relieved TNF-α induced inhibition of cell adhesion and proliferation. We identified a novel TNF-α/HIF-1α/VASP axis in which HIF-1α acts downstream of TNF-α to inhibit VASP expression and modulate the adhesion and proliferation of breast cancer cells. These data provide new insight into the potential anti-tumor effects of TNF-α.     

Century-Scale Responses of Ecosystem Carbon Storage and Flux to Multiple Environmental Changes in the Southern United States

Terrestrial ecosystems in the southern United States (SUS) have experienced a complex set of changes in climate, atmospheric CO₂ concentration, tropospheric ozone (O₃), nitrogen (N) deposition, and land-use and land-cover change (LULCC) during the past century. Although each of these factors has received attention for its alterations on ecosystem carbon (C) dynamics, their combined effects and relative contributions are still not well understood. By using the Dynamic Land Ecosystem Model (DLEM) in combination with spatially explicit, long-term historical data series on multiple environmental factors, we examined the century-scale responses of ecosystem C storage and flux to multiple environmental changes in the SUS. The results indicated that multiple environmental changes shifted SUS ecosystems from a C source of 1.20?±?0.56?Pg (1?Pg?=?10¹⁵?g) during the period 1895 to 1950, to a C sink of 2.00?±?0.94?Pg during the period 1951 to 2007. Over the entire period spanning 1895–2007, SUS ecosystems were a net C sink of 0.80?±?0.38?Pg. The C sink was primarily due to an increase in the vegetation C pool, whereas the soil C pool decreased during the study period. The spatiotemporal changes of C storage were caused by changes in multiple environmental factors. Among the five factors examined (climate, LULCC, N deposition, atmospheric CO₂, and tropospheric O₃), elevated atmospheric CO₂ concentration was the largest contributor to C sequestration, followed by N deposition. LULCC, climate, and tropospheric O₃ concentration contributed to C losses during the study period. The SUS ecosystem C sink was largely the result of interactive effects among multiple environmental factors, particularly atmospheric N input and atmospheric CO_2.     

Diterpenoids from Wedelia prostrata and Their Derivatives and Cytotoxic Activities

One new ent‐kaurane diterpenoid, 11β,16α‐dihydroxy‐ent‐kauran‐19‐oic acid ( 1 ), together with eight known analogues 2 – 9 were isolated from the aerial parts of Wedelia prostrata. One of the acidic diterpenoids, kaurenoic acid ( 3 ), was converted to seven derivatives, 10 – 16 . All compounds were evaluated for their cytotoxic activity in vitro against human leukemia (K562), liver (HepG‐2), and stomach (SGC‐7901) cancer cell lines. Only four kaurenoic acid derivatives, 13 – 16 , with 15‐keto and substitutions at C(19) position, exhibited notable cytotoxic activities on these tumor cell lines with IC₅₀ value ranging from 0.05 to 3.71 μm . Compounds 10 – 12 , with oxime on C(15) showed moderate inhibitory effects and compounds 1 – 9 showed no cytotoxicities on them. Structure–activity relationships were also discussed based on the experimental data obtained. The known derivative, 15‐oxokaurenoic acid 4‐piperdin‐1‐ylbutyl ester ( 17 ), induced typical apoptotic cell death in colon SW480 cells upon evaluation of the apoptosis‐inducing activity by flow‐cytometric analysis.     

Distinct Promoters Affect Pyrroloquinoline Quinone Production in Recombinant Escherichia coli and Klebsiella pneumoniae

Pyrroloquinoline quinone (PQQ) is a versatile quinone cofactor participating in numerous biological processes. Klebsiella pneumoniae can naturally synthesize PQQ for harboring intact PQQ synthesis genes. Previous metabolic engineering of K. pneumoniae failed to overproduce PQQ due to the employment of strong promoter in expression vector. Here we report that a moderate rather than strong promoter is efficient for PQQ production. To screen an appropriate promoter, a total of four distinct promoters—lac promoter, pk promoter of glycerol dehydratase gene (dhaB1), promoter of kanamycin resistance gene, and T7 promoter (as the control)—were individually used for overexpressing the endogenous PQQ genes in K. pneumoniae along with heterologous expression in Escherichia coli. We found that all recombinant K. pneumoniae strains produced more PQQ than recombinant E. coli strains that carried corresponding vectors, indicating that K. pneumoniae is superior to E. coli for the production of PQQ. Particularly, the recombinant K. pneumoniae recruiting the promoter of kanamycin resistance gene produced the highest PQQ (1,700 nmol), revealing that a moderate rather than strong promoter is efficient for PQQ production. Furthermore, PQQ production was roughly proportional to glucose concentration increasing from 0.5 to 1.5 g/L, implying the synergism between PQQ biosynthesis and glucose utilization. This study not only provides a feasible strategy for production of PQQ in K. pneumoniae, but also reveals the exquisite synchronization among PQQ biosynthesis, glucose metabolism, and cell proliferation.     

Superior neovascularization and muscle regeneration in ischemic skeletal muscles following VEGF gene transfer by rAAV1 pseudotyped vectors

Recombinant adeno-associated virus serotype 2 (rAAV2) vector has been widely employed for gene therapy. Recent progress suggests that the new serotypes of AAV showed a better performance than did AAV2 in normal tissues. Here, we evaluate the potential role of human vascular endothelial growth factor (VEGF) gene transfer using rAAV vector pseudotyped with serotype 1 capsid proteins (rAAV1) in the treatment of muscle ischemia. In ischemic skeletal muscles, the rAAV1-LacZ vector allowed higher level, broader distribution, and long-lasting gene expression compared with the rAAV2-LacZ vector. Muscle VEGF165 production following the rAAV1-VEGF165 vector injection was 5-10 times higher than that following the rAAV2-VEGF165 vector injection. VEGF165 production mediated by the rAAV1-VEGF165 vector stimulated a large set of neovascularization with relatively mature vascular structures and enhanced muscle regeneration in the ischemic skeletal muscles. Thus, the rAAV1-VEGF165 vector mediated gene transfer may be a therapeutic approach to peripheral vascular diseases.     

Effects of endogenous beta-amyloid overproduction on tau phosphorylation in cell culture

Alzheimer's disease is characterized by beta-amyloid (Abeta) overproduction and tau hyperphosphorylation. Recent studies have shown that synthetic Abeta promotes tau phosphorylation in vitro. However, whether endogenously overproduced Abeta promotes tau phosphorylation and the underlying mechanisms remain unknown. Here, we used mouse neuroblastoma N2a stably expressing wild-type amyloid precursor protein (APPwt) or the Swedish mutant APP (APPswe) to determine the alterations of phosphorylated tau and the related protein kinases. We found that phosphorylation of tau at paired helical filament (PHF)-1, pSer396 and pThr231 epitopes was significantly increased in cells transfected with APPwt and APPswe, which produced higher levels of Abeta than cells transfected with vector or amyloid precursor-like protein 1. The activity of glycogen synthase kinase-3 (GSK-3) was up-regulated with a concomitant reduction in the inhibitory phosphorylation of GSK-3 at its N-terminal Ser9 residue. In contrast, the activity of cyclin-dependent kinase-5 (CDK-5) and protein kinase C (PKC) was down-regulated. Inhibition of GSK-3 by LiCl, but not inhibition of CDK-5 by roscovitine, arrested Abeta secretion and tau phosphorylation. Inhibition of PKC by GF-109203X activated GSK-3, whereas activation of PKC by phorbol-12,13-dibutyrate inhibited GSK-3. These results suggest that endogenously overproduced Abeta induces increased tau phosphorylation through activation of GSK-3, and that inactivation of PKC is at least one of the mechanisms involved in GSK-3 activation.