植物细胞壁伸展测定仪在蚕豆扩张蛋白特性研究中的应用

扩张蛋白(expansin)在细胞扩张和果实成熟中起着极为重要的作用。植物细胞壁伸展测定仪是研究扩张蛋白必不可少的仪器。为此以电涡流传感器为核心部件装配了一种具有结构简单、操作方便和测量准确等优点的新型测定仪,并利用该仪器研究了蚕豆(Vicia faba)扩张蛋白的特性。结果表明蚕豆根、茎、上胚轴和成熟叶片中均存在扩张蛋白,而且叶片和幼根的扩张蛋白活性最强;免疫印迹证实在蚕豆根、茎、上胚轴和成熟叶片中确实存在扩张蛋白。以上结果说明本仪器灵敏且可靠,用此仪器首次发现在成熟叶片中存在扩张蛋白。     

Microtubule-active agents modify nitric oxide production in pulmonary artery endothelial cells

The effects of specific microtubule-active agents on nitric oxide (NO) production were examined in pulmonary artery endothelial cells (PAEC). PAEC were incubated with taxol, which stabilizes microtubules, or nocodazole, which disrupts microtubules, or both for 2-4 h. We then examined NO production, endothelial NO synthase (eNOS) activity, and eNOS association with heat shock protein (HSP) 90. Incubation of PAEC with taxol (15 microM) for 2-4 h resulted in an increase in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. Incubation of PAEC with nocodazole (50 microM) for 2-4 h induced a decrease in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. The presence of taxol in the culture medium prevented the effects of nocodazole on NO production and eNOS activity in PAEC. Geldanamycin, a HSP90 inhibitor, prevented the taxol-induced increase in eNOS activity. Taxol and nocodazole did not affect eNOS, HSP90, and tubulin protein contents in PAEC, as detected using Western blot analysis. These results indicate that the polymerization state of the microtubule cytoskeleton regulates NO production and eNOS activity in PAEC. The changes in eNOS activity induced by modification of microtubules are due, at least in part, to the altered binding of HSP90 to eNOS protein.     

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.     

Rumen ciliated protozoan fauna of the yak (Bos grunniens) in China with the description of Entodinium monuo n. sp

Rumen ciliate species composition was surveyed in domestic yaks kept in Tibet, Sichuan, and Inner Mongolia, China. Twelve genera including 36 species with 18 formae were identified. The species compositions were slightly different among the three areas: yaks in Tibet had the simplest fauna, in contrast, the fauna of yaks in Inner Mongolia were the most abundant and similar to those found in the cattle kept in the same area. This suggests that the rumen ciliate composition of yaks is affected by that of cattle kept together or in proximity. A new species belonging to the genus Entodinium, Entodinium monuo n. sp., was recognized from the yaks in all areas. This new species was common in the yaks but was not detected in the cattle fed near yaks in Inner Mongolia. There was a similar generic ciliate composition in yaks kept in respective areas. Entodinium was the most predominate ciliate (51.9-61.0%) with total ciliate densities estimated as 10(5)/ml per yak.     

Influence of Atg5 Mutation in SLE Depends on Functional IL-10 Genotype

Increasing evidence supports the involvement of autophagy in the etiopathology of autoimmune diseases. Despite the identification of autophagy-related protein (Atg)-5 as one of the susceptibility loci in systemic Lupus erythematosus (SLE), the consequences of the carriage of these mutations for patients remain unclear. The present work analyzed the association of Atg5 rs573775 single nucleotide polymorphism (SNP) with SLE susceptibility, IFNα, TNFα and IL-10 serum levels, and clinical features, in 115 patients and 170 healthy individuals. Patients who where carriers of the rs573775 T* minor allele presented lower IFNα levels than those with the wild genotype, whereas the opposite result was detected for IL-10. Thus, since IL-10 production was regulated by rs1800896 polymorphisms, we evaluated the effect of this Atg5 mutation in genetically high and low IL-10 producers. Interestingly, we found that the rs573775 T* allele was a risk factor for SLE in carriers of the high IL-10 producer genotype, but not among genetically low producers. Moreover, IL-10 genotype influences SLE features in patients presenting the Atg5 mutated allele. Specifically, carriage of the rs573775 T* allele led to IL-10 upregulation, reduced IFNα and TNFα production and a low frequency of cytopenia in patients with the high IL-10 producer genotype, whereas patients with the same Atg5 allele that were low IL-10 producers presented reduced amounts of all these cytokines, had a lower prevalence of anti-dsDNA antibodies and the latest onset age. In conclusion, the Atg5 rs573775 T* allele seems to influence SLE susceptibility, cytokine production and disease features depending on other factors such as functional IL-10 genotype.     

Identification and functional characterization of phenylglycine biosynthetic genes involved in pristinamycin biosynthesis in Streptomyces pristinaespiralis

Pristinamycin I (PI), a streptogramin type B antibiotic produced by Streptomyces pristinaespiralis, contains the aproteinogenic amino acid l-phenylglycine. Recent sequence analysis led to the identification of a set of putative phenylglycine biosynthetic genes. Successive inactivation of the individual genes resulted in a loss of PI production. Production was restored by supplementation with externally added l-phenylglycine, which demonstrates that these genes are involved in phenylglycine biosynthesis and thus probably disclosing the last essential pristinamycin biosynthetic genes. Finally, a putative pathway for phenylglycine synthesis is proposed.     

Divergent kinase signaling mediates agonist-induced phosphorylation of phosphatase inhibitory proteins PHI-1 and CPI-17 in vascular smooth muscle cells

Phosphatase holoenzyme inhibitor (PHI)-1 is one of the newest members of the family of protein phosphatase inhibitor proteins. In isolated enzyme systems, several kinases, including PKC and rho kinase (ROCK), have been shown to phosphorylate PHI-1. However, it is largely unknown whether PHI-1 is phosphorylated in response to agonist stimulation in intact cells. We investigated this question in primary cultured rat aortic vascular smooth muscle cells (VSMCs). Using two-dimensional polyacrylamide gel electrophoresis and immunoblot, we found that there are two major PHI-1 spots under resting conditions: a minor spot with an acidic isoelectric point (pI) and a major spot with a more alkaline pI. Interestingly, U-46619, a G protein-coupled receptor agonist, caused a significant increase in the acidic spot, suggesting that it may represent a phosphorylated form of PHI-1. This was confirmed by phosphatase treatment and by a specific phospho-PHI-1 antibody. Furthermore, we found that angiotensin II, thrombin, and U-46619 increased phosphorylated PHI-1 from 9% of total PHI-1 in resting cells to 18%, 18%, and 30%, respectively. We also found that inhibition of ROCK by Y-27632 or H-1152 selectively diminished U-46619-induced CPI-17 phosphorylation, whereas it did not affect PHI-1 phosphorylation. Activation of ROCK by expressing V14RhoA selectively induced CPI-17 phosphorylation without affecting PHI-1 phosphorylation. In contrast, inhibition of PKC by GF-109203X or by PKC downregulation selectively diminished U-46619-induced PHI-1 phosphorylation without significantly affecting U-46619-induced CPI-17 phosphorylation. Activating PKC by PMA induced PHI-1 phosphorylation. Together, our results show for the first time that agonist induces PHI-1 phosphorylation in VSMCs and divergent kinase signaling couples agonist stimulation to PHI-1 and CPI-17 phosphorylation. signal transduction; myosin phosphatase holoenzyme inhibitor 1; protein kinase C     

南方红豆杉叶精油化学成分的研究

采用水蒸汽蒸馏法提取南方红豆杉叶的精油,通过色谱－质谱－计算机联用技术分析了精油的化学成分。从中初步鉴定出２６个化合物,占精油总量的９６％,主要成分是：棕榈酸（３５．６６％）,９－十六碳烯酸９－十六碳稀酯（１１．２８％）,３－辛醇（４．４７％）,１－苯－１,２－丙二酮（４．３％）,Ｎ－苯基－１－萘胺（３．５７％）,１,２－苯二羧酸（２－甲基丙基）二酯（３．５７％）,６,１０－二甲基－５,９－十一双     

High Genetic Differentiation of Hippophae rhamnoides ssp. yunnanensis (Elaeagnaceae), a Plant Endemic to the Qinghai-Tibet Plateau

RAPD markers were used to detect genetic diversity and population genetic differentiation of Hippophae rhamnoides ssp. yunnanensis, a sea buckthorn endemic to the Qinghai-Tibet plateau. The genetic parameters of percentage of polymorphic bands (92.86%), Nei’s gene diversity (h, 0.255), and Shannon’s index (I, 0.397) indicated high genetic diversity in this subspecies. The subpopulation differentiation suggested that 45.9% of genetic variation was among populations. High genetic differentiation among populations was also detected using AMOVA (47.02%). The main factors responsible for high genetic differentiation are probably related to natural geographic barriers among populations, gene drift, and limited gene flow caused by restricted pollen flow and seed flow. A Mantel test indicated that geographic distances were significantly correlated with genetic distances. The UPGMA phenogram based on Nei’s unbiased genetic distances and the result of three-dimensional model plots performed by principal coordinate analysis also supported the correlation. Altitude, however, did not have any clear effect on genetic differentiation.