瘦素诱导体外培养大鼠脂肪间充质干细胞凋亡

为观察瘦素诱导体外培养大鼠脂肪间充质干细胞凋亡的作用, 采用胶原酶消化法分离培养大鼠附睾脂肪垫间充质干细胞, 第3代细胞用于实验。细胞免疫荧光化学方法鉴定CD105、Vimentin表达阳性率约80%以上, 10-6 mol/L的瘦素作用细胞48 h、72 h后激光共聚焦显微镜观察分别可见早期及中晚期特征表现; 0 mol/L、10-8 mol/L、10-7 mol/L、10-6 mol/L瘦素分别作用于细胞48 h后, 应用AnnexinⅤ/PI双染色法流式细胞仪检测早期凋亡率分别为2.50%±0.72%、6.78%±1.99%、11.99%±1.58%、17.93%±4.82% (P<0.05); 随着瘦素浓度的增加和作用时间的延长, Caspase-3的活性逐渐增高, 至48 h时达到高峰。说明瘦素可以直接诱导脂肪间充质干细胞凋亡, 从数量上减少脂肪组织的含量。     

Store-dependent and -independent modes regulating Ca2+ release-activated Ca2+ channel activity of human Orai1 and Orai3

We evaluated currents induced by expression of human homologs of Orai together with STIM1 in human embryonic kidney cells. When co-expressed with STIM1, Orai1 induced a large inwardly rectifying Ca(2+)-selective current with Ca(2+)-induced slow inactivation. A point mutation of Orai1 (E106D) altered the ion selectivity of the induced Ca(2+) release-activated Ca(2+) (CRAC)-like current while retaining an inwardly rectifying I-V characteristic. Expression of the C-terminal portion of STIM1 with Orai1 was sufficient to generate CRAC current without store depletion. 2-APB activated a large relatively nonselective current in STIM1 and Orai3 co-expressing cells. 2-APB also induced Ca(2+) influx in Orai3-expressing cells without store depletion or co-expression of STIM1. The Orai3 current induced by 2-APB exhibited outward rectification and an inward component representing a mixed calcium and monovalent current. A pore mutant of Orai3 inhibited store-operated Ca(2+) entry and did not carry significant current in response to either store depletion or addition of 2-APB. Analysis of a series of Orai1-3 chimeras revealed the structural determinant responsible for 2-APB-induced current within the sequence from the second to third transmembrane segment of Orai3. The Orai3 current induced by 2-APB may reflect a store-independent mode of CRAC channel activation that opens a relatively nonselective cation pore.     

Polymorphisms of 5,10-methylenetetralydrofolate reductase (MTHFR), fruit and vegetable intake, and the risk of stomach cancer

Stomach cancer is a serious public health problem in China. 5,10-Methylenetetralydrofolate reductase (MTHFR) may be involved in both DNA methylation and DNA synthesis. Folate deficiency is associated with cancer risk that may be modulated by a genetic variation in the MTHFR gene in folate metabolism. The main goal of this study was to evaluate the association between polymorphisms of the MTHFR gene and the risk of stomach cancer. This study also explored the modification effects of fruit and vegetable intake (one of the main constituents is folate) on the risk of this disease. A population-based case-control study was conducted in Taixing, China, consisting of 206 newly diagnosed cases with primary stomach cancer and 415 healthy population controls. Polymorphisms of MTHFR C677T and A1298C were assayed by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) techniques. The data were analysed using the logistic regression model. No obvious association between the MTHFR A1298C polymorphism and the risk of stomach cancer was observed in this study. The frequencies of 677 C/C, C/T, and T/T were 34.5, 50.9, and 14.6%, respectively, in controls. The frequency of the MTHFR 677 wild homozygotic genotype was 25.8% in cases, which was lower than that in controls (34.5%). The adjusted odds ratio (OR) for the MTHFR 677 any T genotype was 2.05 (95% confidence interval (CI), 1.26-3.34) when compared with the C/C genotype. In the low fruit and vegetable intake group an increasing trend was observed with the T allele exposure, p=0.0056. The adjusted ORs were 1.68 (95% CI = 0.86-3.29) for the C/T genotype and 3.58 (95% CI = 1.46-8.75) for the T/T genotype, respectively. The MTHFR 677 any T genotype was associated with an increased risk of primary stomach cancer among the Chinese population. Folate deficiency might modify the MTHFR gene polymorphism and influence the risk of stomach cancer.     

Fumonisins production by Fusarium proliferatum strains isolated from asparagus crown

The present work deals with the capability for producing fumonisin by Fusarium proliferatum strains isolated from asparagus in China. Fifty of F. proliferatum strains were randomly selected and incubated on cultures of maize grain and asparagus spear, respectively. Fumonisin levels (FB₁ and FB₂) were determined by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results showed that all 50 strains produced fumonisins in maize culture within a wide range of concentrations, 10–11,499 μg/g and 2–6,598 μg/g for FB₁ and FB₂, respectively. On culture of asparagus spear,48 strains (96%) produced fumonisins in the range 0.2–781.6 μg/g and no detected to 40.3 μg/g for FB₁ and FB₂, respectively. All of F. proliferatum strains produced much higher levels of FB₁, FB₂ and total fumonisins (FB₁ + FB₂) in maize grain culture than in asparagus spear culture. Meanwhile, fumonisin B₃ (FB₃) was identified in all maize culture extracts and most of asparagus spear culture extracts. This is the first study carried out the fumonisin-producing ability of F. proliferatum strains isolated from asparagus in China. The information obtained is useful for assessing the risk of fumonisins contamination in asparagus spear. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Selection of aptamers against inactive Vibrio alginolyticus and application in a qualitative detection assay

Aptamers against inactive Vibrio alginolyticus were selected from an 82-nt ssDNA random library by systematic evolution of ligands by exponential enrichment. After 15 rounds of selection, the final pool of aptamers was highly specific for inactivated V. alginolyticus and had a dissociation constant of 27.5 ± 9.2 nM. Using these aptamers and PCR, V. alginolyticus could be detected at 100 cells/ml. Sequencing of the final pool of aptamers revealed that some sequences, termed high-frequency aptamers, appeared more than once; these may be of practical application. All sequences obtained were divided into nine families according to their homology tree, some conserved sequences were also found in each of the six families. One sequence was found in significant proportions of the aptamers, suggesting that this conserved sequence might be important for forming the three-dimensional aptamer structure.     

Nitric Oxide Inhibits Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia by Increasing the Ubiquitination and Degradation of UbcH10

Nitric oxide (NO) limits formation of neointimal hyperplasia in animal models of arterial injury in large part by inhibiting vascular smooth muscle cell (VSMC) proliferation through cell cycle arrest. The ubiquitin-conjugating enzyme UbcH10 is responsible for ubiquitinating cell cycle proteins for proper exit from mitosis. We hypothesize that NO prevents VSMC proliferation, and hence neointimal hyperplasia, by decreasing levels of UbcH10. Western blotting and immunofluorescent staining showed that NO reduced UbcH10 levels in a concentration-dependent manner in VSMC harvested from the abdominal aortas of Sprague-Dawley rats. Treatment with NO or siRNA to UbcH10 decreased both UbcH10 levels and VSMC proliferation (P<0.001), while increasing UbcH10 levels by plasmid transfection or angiotensin II stimulation increased VSMC proliferation to 150% (P=0.008) and 212% (P=0.002) of control, respectively. Immunofluorescent staining of balloon-injured rat carotid arteries showed a ~4-fold increase in UbcH10 levels, which was profoundly decreased following treatment with NO. Western blotting of carotid artery lysates showed no UbcH10 in uninjured vessels, a substantial increase in the injury alone group, and a significant decrease in the injury+NO group (~3-fold reduction versus injury alone). Importantly, in vitro and in vivo, a marked increase in polyubiquitinated UbcH10 was observed in the NO-treated VSMC and carotid arteries, respectively, indicating that NO may be decreasing unmodified UbcH10 levels by increasing its ubiquitination. Central to our hypothesis, we report that NO decreases UbcH10 levels in VSMC in vitro and following arterial injury in vivo in association with increasing polyubiquitinated-UbcH10 levels. These changes in UbcH10 levels correlate with VSMC proliferation and neointimal hyperplasia, making UbcH10 a promising therapeutic target for inhibiting this proliferative disease.     

Amplification, analysis and chromosome mapping of novel homeobox-containing and homeobox-flanking sequences in rice

Homeobox genes, widely distributed among animal and plant kingdoms, play an important role in developmental process. Several homeobox conserved fragments were amplified by PCR and the flanking regions were also obtained by an LM-PCR procedure. Sequencing and Southern analysis showed that they belong to a homeobox gene family of rice. Six homeobox-containing fragments were mapped on the molecular linkage map of rice. They were located on chromosomes 3, 4 and 7 respectively. It is noteworthy that there are 4 homeobox fragments located on rice chromosome 3 and the result is also consistent with the comparative genomics between rice and maize.     

铁蛋白基因表达对烟草耐低铁能力的影响

铁是植物生长发育的必需元素。由于土壤中的三价铁离子不能被植物直接利用, 使一些植物经常表现出缺铁症状。为探讨利用铁蛋白基因提高植物耐低铁胁迫的作用, 利用农杆菌介导法将大豆铁蛋白基因SoyFer1和内源反义铁蛋白基因NtFer2的cDNA分别导入烟草基因组, 采集转基因烟草种子。对T1转基因烟草的卡那霉素抗性分析表明, 整合到烟草基因组的外源基因多为单拷贝基因, 也有少数为多拷贝基因。对具有卡那霉素抗性的转基因植株进行PCR检测和Northern杂交分析表明, 外源基因已整合到烟草基因组中, 并且得到了正确表达。将转基因株系移栽到铁离子浓度不同的培养基中生长2个月后进行比较表明, 转大豆铁蛋白基因烟草株系的生长量明显高于非转基因烟草株系, 而转内源反义铁蛋白基因烟草株系的生长量则明显低于非转基因烟草株系。转大豆铁蛋白基因和转内源反义铁蛋白基因烟草株系的叶绿素含量、丙二醛(MDA)含量和过氧化物酶(POD)活性等生理性状也发生了明显变化, 表现为转大豆铁蛋白基因株系的叶绿素含量明显增加, POD活性明显增强, MDA含量明显降低; 而转内源反义铁蛋白基因株系的叶绿素含量、POD活性和MDA含量等则表现为与转大豆铁蛋白基因株系的相反。铁蛋白过量表达提高了烟草耐低铁能力, 而铁蛋白抑制表达则降低了烟草耐低铁能力。