A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase from Rahnella aquatilis with Significantly Reduced Glyphosate Sensitivity

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroA(R.aquatilis), was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroA(E.coli)), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroA(R.aquatilis) were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroA(E.coli). To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious.     

游泳运动对低氧性肺动脉高压大鼠肺组织apelin及其受体表达的影响

目的:探讨游泳运动对大鼠肺组织新的小分子活性肽apelin及其受体(APJ)表达的影响。方法:45只雄性大鼠随机分成三组:正常对照组、低氧组(七周)和游泳组(低氧+游泳锻炼七周组,低氧3周后,于每天入低氧舱前行无负重游泳运动60 min,每天1次)。七周后测定各组大鼠平均肺动脉压(mPAP)、右心室与左心室加室间隔的重量比[RV/(LV+S)]、肺细小动脉管壁面积/管总面积(WA/TA)、管腔面积/管总面积(CA/TA)及中膜厚度(PAMT)。免疫蛋白印迹与免疫组化法测定肺组织apelin/APJ的蛋白表达。结果:①低氧组mPAP和RV/(LV+S)比正常对照组分别高73.6%和31.2%(P均<0.01),而游泳组比低氧组分别低21.1%和8.9%(P均<0.05)。②低氧组WA/TA和PAMT较正常对照组分别高70.8%和102%,而游泳组较低氧组分别低24.8%和40.1%(P均<0.01)。低氧组CA/TA较正常对照组低15.1%,而游泳组较低氧组高10.3%(P均<0.01)。③低氧组肺组织apelin蛋白表达较正常对照组上调374%(P<0.01),而APJ蛋白表达下调87.1%(P均<0.01);游泳组肺组织apelin蛋白表达较低氧组下调48%,而APJ蛋白表达上调287%(P均<0.01)。④apelin蛋白主要在血管外膜及炎症细胞胞浆内表达,APJ蛋白主要在血管内膜、外膜及炎症细胞上表达。结论:游泳运动减缓肺动脉高压和肺血管重塑作用可能与调节肺组织apelin/APJ系统的表达有关。     

B7-H3 is expressed in human hepatocellular carcinoma and is associated with tumor aggressiveness and postoperative recurrence

B7-H3, a novel B7 family member, positively or negatively regulates T-cell responses. We investigated the clinical relevance and prognostic significance of B7-H3 in hepatocellular carcinoma (HCC). Western blotting showed B7-H3 upregulation in 17 of 24 (70.8 %) HCC tissues compared with nontumor liver tissues (p = 0.028). B7-H3 immunostaining on tissue microarrays containing 240 HCC patient samples indicated that 225 (93.8 %) tumors had aberrant B7-H3 expression, with strong intensity in 79 (32.9 %) cases, whereas B7-H3 expression in peritumor liver cells was weak in most cases (226; 94.2 %). Notably, patients with high/moderate tumor cell B7-H3 expression showed significantly poorer survival (p = 0.009) and increased recurrence (p = 0.002). After multivariable adjustment, high/moderate B7-H3 expression remained significant for an increased risk of recurrence (hazard ratio = 1.79; 95 % confidence interval = 1.19–2.70; p = 0.005). B7-H3 expression correlated with invasive phenotypes like vascular invasion and advanced tumor stage, and the metastatic potential of HCC cell lines. Flow cytometry showed that B7-H3 expression is inversely correlated with proliferation and interferon-γ production by infiltrating T cells. Interferon-γ stimulation significantly upregulated B7-H3 expression in HCC cells in vitro, implicating B7-H3 expression as a feedback mechanism to evade anti-tumor immunity. Importantly, the prognostic value of B7-H3 expression was validated in an independent cohort of 206 HCC patients. Collectively, our data suggest that B7-H3 was abundantly expressed in HCC and was associated with adverse clinicopathologic features and poor outcome. Thus, B7-H3 represents an attractive target for diagnostic and therapeutic manipulation in human HCC.     

Isolation and characterization of polymorphic microsatellite loci from black sea bass (Centropristis striata) and cross-species amplification

Black sea bass (Centropristis striata) is an economically important serranid species. A number of 39 microsatellite loci were isolated from two enriched genomic library of C. striata. Eleven of these loci were polymorphic in a test population with alleles per locus ranging from 3 to 8, and observed and expected heterozygosities per locus from 0.26 to 1.00 and from 0.61 to 0.84, respectively. Four loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of C. striata and other related species.     

Immobilized enzymatic fluorescence capillary biosensor for determination of sulfated bile acid in urine

The authors have proposed an immobilized enzymatic fluorescence capillary biosensor (SBAs-IE-FCBS) for the determination of sulfated bile acids (SBAs). The reaction principle of the biosensor is that under the catalysis of the bile acid sulfate sulfatase (BSS) and beta-hydroxysteroid dehydrogenase (beta-HSD) immobilized on inner surface of a medical capillary, SBAs desulfates to 3beta-hydroxyl bile acids, then the latter reacts with nicotinamide adenine dinucleotide (NAD(+)), and is converted into 3-ketosteroid; meanwhile, NAD(+) is converted to reduced nicotinamide adenine dinucleotide (NADH). NADH continuously reacts with 1-methoxy-5-methylphenazinium methyl sulfate (1-MPMS) and is converted into NAD(+) circularly and 1-MPMSH(2). Finally resazurin is reduced into resorufin by 1-MPMSH(2), the formed resorufin (lambda(ex)/lambda(em): 540 nm/580 nm) is used for quantifying the concentration of SBAs. Optimized conditions being suitable with the biosensor are as follows: the concentrations of BSS and beta-HSD used for the immobilization all are 5 kUL(-1); the concentrations of 1-MPMS and resazurin all are 25 micromolL(-1); the concentrations of Tris-HCl buffer and NAD(+) are 100 and 400 micromolL(-1), respectively; total volume of the enzyme, reagent and sample is only 18 microL per time for determining; the reaction temperature is 37 degrees C; the reaction time is 15min. The concentration of SBAs is directly proportional to the fluorescence intensity of the biosensor measured from 0.5 to 5.0 micromolL(-1). The relative standard deviation is less than 3.4%, and the detection limit was 0.16 micromolL(-1). The recoveries are in the range 95.5-106%. This SBA-IE-FCBS can be used for quantifying SBAs in urine to diagnose and judge hepatobiliary diseases, etc.     

There is more to tomato fruit colour than candidate carotenoid genes

Determining gene sequences responsible for complex phenotypes has remained a major objective in modern biology. The candidate gene approach is attempting to link, through mapping analysis, sequences that have a known functional role in the measured phenotype with quantitative trait loci (QTL) that are responsible for the studied variation. To explore the potential of the candidate approach for complex traits we conducted a mapping analysis of QTL for the intensity of the red colour of the tomato fruit (mainly lycopene) and for probes associated with the well-characterized carotenoid biosynthesis pathway. Seventy-five tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii delimited 107 marker-defined mapping bins. Three of the bins resolved known qualitative colour mutations for yellow (r) and orange (B and Del) fruits resulting from variation in specific carotenoid biosynthesis genes. Based on trials in different environments, 16 QTL that modified the intensity of the red colour of ripe fruit were assigned to bins. Candidate sequences associated with the carotenoid biosynthesis pathway were mapped to 23 loci. Only five of the QTL co-segregated with the same bins that contained candidate genes - a number that is expected by chance alone. Furthermore, similar map location of a QTL and a candidate is far from a direct causative relationship between a gene and a phenotype. This study highlights the wealth and complexity of the variation present in the genus Lycopersicon that could be employed for basic research and genetic improvement of fruit colour in tomato.     

红火蚁病原真菌分离鉴定及致病性研究

红火蚁Solenopsis invicta是世界上最具破坏力和威胁性的入侵检疫害虫之一,对农林业生产安全、公共设施以及人体健康都造成了巨大影响。为筛选对红火蚁有致病性的病原真菌,本研究通过组织分离法对自然罹病的红火蚁进行致病菌分离,结合病原形态学及分子生物学的方法对病原真菌进行鉴定并采用喷雾法测定了病原真菌对红火蚁的致病力。结果表明：从罹病红火蚁上分离出3株病原真菌HHYSFJ01、HHYSFJ02和HHYSFJ05,分别为红绶曲霉Aspergillus nomius、淡紫拟青霉Purpureocillium lilacinum和球孢白僵菌Beauveria bassiana。致病性测定,这3株病原菌对红火蚁均具有较强的致病性,且3株病原菌致病性间差异显著。经1.0×10^8孢子/mL孢子悬浮液喷雾处理红火蚁10 d后,球孢白僵菌菌株HHYSFJ05的致病性最强,红火蚁工蚁的校正死亡率为84.71%,LT50为4.67 d;红绶曲霉菌株HHYSFJ01和淡紫拟青霉菌株HHYSFJ02次之,红火蚁工蚁的校正死亡率分别为78.82%和73.29%,LT50分别为4.99 d和5.20 d。     

鲤微卫星引物对麦穗鱼的适用性初步研究

麦穗鱼（Pseudorasbora　parva　Temminck　et　Schlegel）是淡水中广泛分布的小型鱼类,属鲤科（Cyprinidae）、鲍亚科（Gobioninae）、麦穗鱼属（Pseudorasbora　Bleeker）。常见于中国东部地区各大水系,朝鲜、日本也有分布。开展小型鱼类种群遗传多样性研究,能为合理开发利用小型鱼类资源,发展鱼食性鱼类增养殖,优化湖泊渔业结构提供重要的基础资料。     

Alteration of Whole Leaf Protein of a Temperature sensitive Chlorophyll Deficient Rice Mutant During the Bleaching Process

l103s was a rice ( Oryza sativa L. ) mutant whose leaves would mm into chimerical chlorophylldeficient when treated with varied temperature (high - low - high). The alteration of whole leaf protein was studied with two-dimensional electrophoresis during the bleaching process. No changes of large subunit and small subunit of Rubisco had been detected in the bleached tissues of 1103s, but a 51 kD (PI =-4.5) special polypeptide-P1 disappeared in the white area during the bleaching process. P1 polypeptide was also detected in those tissues which remained green on the chlorophyll deficient leaves, although its amount apparenfiy decreased. In the tissue of low-temperature treated 1103s and l103s × 8902s, the normal expression of P1 polypeptide had been detected. The data suggest that P1 is an important chlorophyll-synthesis related down stream functional protein. In 1103s, it is regulated by some yet-tmknown temperature-sensitive mechanisms.