细菌脂多糖诱导人单核细胞株对细菌脂蛋白的耐受性及其与肌动蛋白骨架关系

细菌脂多糖(LPS)可诱导宿主对LPS的耐受,但对细菌脂蛋白(BLP)是否存在交叉耐受,目前报道不一。采用人单核细胞株(THP-1),建立小剂量LPS诱导THP-1对LPS耐受的细胞模型;观察细胞肌动蛋白骨架、炎症因子TNF-α、IL-1β、IL-6的浓度及NF-κB的DNA结合活力的变化情况;探讨BLP交叉耐受及细胞骨架在其中的作用。结果显示,THP-1细胞经小剂量(10ng/ml)LPS、大剂量(100ng/ml)LPS或BLP刺激后,细胞形态严重变形,肌动蛋白重组,细胞周边肌动蛋白丝带消失,出现明显的肌动蛋白收缩团块及伪足,细胞核内NF-κB的DNA结合活性显著升高,培养上清液中炎症因子(TNF-α、IL-1β及IL-6)的释放显著增加;而小剂量LPS预刺激12h后,再用大剂量的LPS或BLP刺激6h,上述指标明显改善;采用细胞骨架肌动蛋白聚集破坏剂鬼笔环肽预处理后的THP-1细胞,可取消由小剂量LPS诱导的自身耐受及对BLP的交叉耐受;可见,细菌LPS、BLP(100ng/ml)可诱导THP-1细胞肌动蛋白骨架的改变,激活NF-κB信号通路,诱导炎性细胞因子TNF-α、IL-1、IL-6过度释放,激活宿主炎症细胞的炎症反应;而小剂量LPS预刺激后可诱导出THP-1细胞对LPS的自身耐受和对BLP的交叉耐受;细胞骨架肌动蛋白参与了小剂量LPS诱导THP-1细胞对LPS自身耐受和对BLP交叉耐受的形成。     

Culture media conditioned by heat-shocked osteoblasts enhances the osteogenesis of bone marrow-derived mesenchymal stromal cells

Osteogenic cells differentiated from bone marrow-derived mesenchymal stromal cells (MSC) hold much promise in bone tissue engineering and reconstructive surgery. There is a dire need for well-defined and efficient protocols to promote the osteogenesis of ex vivo cultured MSC. Hence, this study investigated whether a combination of chemical stimuli (ascorbic acid, beta-glycerophosphate and dexamethasone) and culture media conditioned by a human foetal osteoblast cell line (hFOB) had any synergistic effect on the osteogenesis of MSC. Conditioned media with or without prior heat shock treatment (42 degrees C for 1 h) of the hFOB cell line, were collected and tested on rabbit MSC cultures, in the presence and absence of chemical stimuli. Osteogenic differentiation of MSC was assessed on both day 14 and 21 of ex vivo culture. The results showed conclusively that conditioned media promoted osteogenesis of MSC, which was further enhanced by prior heat shock-treatment of the hFOB cells, as well as by the presence of chemical stimuli. Among all experimental groups, the combination of culture medium conditioned by heat shocked hFOB cells together with chemical stimuli, exhibited the highest level of calcium mineralization, as assessed by Von Kossa staining. This provides clear evidence of a synergistic effect of conditioned media, heat shock and chemical stimuli. It is hoped that the data may contribute to the development of a more well-defined and efficient in vitro culture protocol to promote the osteogenesis of MSC for both clinical and non-clinical applications.     

Role for Fox-1/Fox-2 in mediating the neuronal pathway of calcitonin/calcitonin gene-related peptide alternative RNA processing

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.     

Nitricoxide synthase-induced oxidative stress in prolonged alcoholic myopathies of rats

Previous studies showed that nitricoxide synthase (NOS) and oxidative stress can induce skeletal muscle atrophy in the muscular dystrophy and inclusion-body myopathy. There is a correlation between NOS and oxidative stress. However, it is not clear, whether there are some changes of the NOS activity in prolonged alcoholic myopathy (PAM), and whether NOS activity has relation to amyotrophy of PAM. We established experimental alcoholic myopathy model of rats by prolonged alcohol intake. We found that there is a reduction in GSH-px (P < 0.05) and an increase of SOD (P < 0.05), MDA (P < 0.05) and iNOS (P < 0.05) in the plantaris of the experimental group by spectrophotometer. In the soleus of the experimental group, except for MDA showed an increase (P < 0.05), the other enzymes showed no obvious difference (P > 0.05). The immunohistochemistry results showed that there was obvious expression of iNOS in the cytoplasm of plantaris in the experimental group and there was no expression of iNOS in the control group. There was a decrease of nNOS expression on the membranes of the plantaris cells in the experimental group by immunofluorescence. Meanwhile, we found the expression of nNOS in some cytoplasm. Our results suggested that NOS might be an important factor during the development of PAM. We could infer that there are some disturbances with regard to output and scavenging of free radical in PAM. Alcohol can induce the oxidative stress reaction and further result in imbalance of the oxidant-antioxidant status in the organism. Haiying Chu is the co-first author.     

<Emphasis Type="Italic">Sporotalea propionica</Emphasis> gen. nov. sp. nov., a hydrogen-oxidizing,oxygen-reducing,propionigenic firmicute from the intestinal tract of a soil-feeding termite

An unusual propionigenic bacterium was isolated from the intestinal tract of the soil-feeding termite Thoracotermes macrothorax. Strain TmPN3 is a motile, long rod that stains gram-positive, but reacts gram-negative in the KOH test. It forms terminal endospores and ferments lactate, glucose, lactose, fructose, and pyruvate to propionate and acetate via the methyl-malonyl-CoA pathway. Propionate and acetate are formed at a ratio of 2:1, typical of most propionigenic bacteria. Under a H₂/CO₂ atmosphere, the fermentation product pattern of glucose, fructose, and pyruvate shifts towards propionate formation at the expense of acetate. Cell suspensions reduce oxygen with lactate, glucose, glycerol, or hydrogen as electron donor. In the presence of oxygen, the product pattern of lactate fermentation shifts from propionate to acetate production. 16S rRNA gene sequence analysis showed that strain TmPN3 is a firmicute that clusters among the Acidaminococcaceae, a subgroup of the Clostridiales comprising obligately anaerobic, often endospore-forming bacteria that possess an outer membrane. Based on phenotypic differences and less than 92% sequence similarity to the 16S rRNA gene sequence of its closest relative, the termite hindgut isolate Acetonema longum, strain TmPN3^T is proposed as the type species of a new genus, Sporotalea propionica gen. nov. sp. nov. (DSM 13327^T, ATCC BAA-626^T).     

Knockout of the gene (<Emphasis Type="Italic">ste15</Emphasis>) encoding a glycosyltransferase and its function in biosynthesis of exopolysaccharide in <Emphasis Type="Italic">Streptomyces</Emphasis> sp. 139

Streptomyces sp. 139 produces a novel exopolysaccharide (EPS) designated Ebosin which has antagonistic activity for IL-1R in vitro and remarkable anti-rheumatic arthritis activity in vivo. We previously identified a ste (Streptomyces eps) gene cluster consisting of 27 ORFs responsible for Ebosin biosynthesis. The gene product of ste15 shows high homology to known glycosyltransferases (GTFs). To elucidate its function in Ebosin biosynthesis, the ste15 gene was knocked out with a double crossover via homologous recombination. Our analysis of monosaccharide composition for EPS-m produced by the mutant strain Streptomyces sp. 139 (ste15 ⁻) showed that glucose was significantly diminished compared to its natural counterpart Ebosin. This derivative of Ebosin lost the antagonistic activity for IL-1R in vitro and its molecular mass was smaller than Ebosin. These results have demonstrated that the ste15 gene codes for a GTF for glucose, which is functionally involved in Ebosin biosynthesis.     

Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future.     

小孢子链格孢endoPG基因核苷酸序列分析及系统发育研究

对供试小孢子链格孢菌株的内聚半乳糖醛酸酶(endoPG)基因进行扩增,大部分菌株都可获得PCR产物。核苷酸和氨基酸序列比较表明:不同种小孢子链格孢endoPG基因核苷酸序列存在明显差异,甚至表现在氨基酸水平,这些差异可以作为一些种如梨黑斑链格孢、长柄链格孢区分的分子性状。利用邻近结合法构建系统发育树,所有菌株被分为8个聚类组。在系统发育树上,链格孢的一些不同分离物被聚在不同组中,而细极链格孢、链格孢的部分菌株、苹果链格孢、柑橘链格孢、粗柠檬褐斑链格孢、橘树链格孢被聚为一组,显示根据形态学特征划分的这些种与分子性状的不一致性。endoPG基因核苷酸序列富于变化,为小孢子链格孢系统发育研究提供了一种有用的手段。     

Imidazole as a catalyst for in vitro refolding of enhanced green fluorescent protein

Imidazole is a reagent widely used in protein purifying processes. Here, we reveal a novel chaperone-like activity for imidazole using enhanced green fluorescent protein (EGFP) as a model protein. Experimental results showed that imidazole acted as an effective catalyst for refolding of the chemically denatured EGFP and suppressor for the heat-induced aggregation of EGFP. The refolding kinetics was determined in real time. Both the recovering yield and refolding rate of denatured EGFP in the presence of imidazole were increased. The studies on elucidating the mechanism show that imidazole may catalyze the prolyl cis/trans isomerization and the possible mechanism was discussed. To our knowledge, there are no data on the effect of imidazole on protein folding. Considering the prolyl isomerization is the rate-limited step for refolding of most proteins and aggregation is a universal serious problem for biotechnology, imidazole thus represents a previous unknown type of protein-folding catalyst.