In vitro development and transfer of resistance to chlortetracycline in Bacillus subtilis

The present criteria and rules controlling the approval of the use of probiotics are limited to antibiotic resistance patterns and the presence of antibiotic resistance genes in bacteria. There is little information available in the literature regarding the risk of the usage of probiotics in the presence of antibiotic pressure. In this study we investigated the development and transfer of antibiotic resistance in Bacillus subtilis selected in vitro by chlortetracycline in a stepwise manner. Bacillus subtilis was exposed to increasing concentrations of chlortetracyclineto induce in vitro resistance to chlortetracycline, and the minimal inhibitory concentrations were determinedfor the mutants. Resistant B. subtilis were conjugated with Escherichia coli NK5449 and Enterococcus faecalis JH2-2 using the filter mating. Three B. subtilis tetracycline resistant mutants (namely, BS-1, BS-2, and BS-3) were derived in vitro. A tetracycline resistant gene, tet (K), was found in the plasmids of BS-1 and BS-2. Three conjugates (BS-1N, BS-2N, and BS-3N) were obtained when the resistant B. subtilis was conjugated with E. coli NK5449. The conjugation frequencies for the BS-1N, BS-2N, and BS-3N conjugates were 4.57×10^?7, 1.4×10^?7, and 1.3×10^?8, respectively. The tet(K) gene was found only in the plasmids of BS-1N. These results indicate that long-term use of probiotics under antibiotic selection pressure could cause antibiotic resistance, and the resistance gene could be transferred to other bacteria. The risk arising from the use of probiotics under antibiotic pressure should be considered in the criteria and rules for the safety assessment of probiotics.     

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]_1/2 at 3.4-5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys³⁶-Cys⁴⁹ and two disulfide bonds formed by two pair of consecutive cysteines, Cys²²-Cys²³ and Cys⁵⁶-Cys⁵⁷, a unique disulfide structure of polypeptide that has not been documented previously.     

人类新基因WDR24的研究初报

WD40家族是一类结构保守、功能复杂的蛋白.目前很多研究显示该家族成员通过参与MAPK信号途径调控细胞内信号转导而影响细胞的基本生命活动.为了鉴定参与细胞生命活动的新基因,运用同源基因克隆法,通过PCR技术扩增获得一个新的人类基因WDR24, 其cDNA全长3 302 bp,2 373 bp长的开放阅读框编码由790个氨基酸残基组成的蛋白质.生物信息学分析表明,WDR24蛋白在进化上高度保守,与其他脊椎动物中的同源蛋白组成了一个功能未知的亚家族.蛋白序列分析显示其中有6个WD40重复序列和1个ERK的停泊位点D-domain.RT-PCR分析表明,该基因在所有被检测的人类胚胎组织中表达.     

5种光合细菌种间原生质体融合及优良农用融合子的筛选鉴定

处于对数生长期的光合细菌球形红假单胞菌(Rhodopseudomonassphaeroides)、沼泽红假单胞菌(Rhodopseudomonaspalustris)、嗜酸红假单胞菌(Rhodopseudomdnasacidophila)、深红红螺菌(Rhodospirarubrum)、万尼氏红微菌(Rhodomocrobiumvannielii),经溶菌酶(3mg/L)处理50min后,获得了它们的菌体形成的原生质体,其再生率分别为80%、71%、82%、61%、74%.取等量的亲本菌株在35%的PEG(MW6000)诱导下两两融合5min,共10种组合.其融合率为球×沼2.5×10-4、球×嗜2.1×10-4、球×深2.0×10-4、球×万2.1×10-4、沼×嗜2.8×10-4、沼×深2.4×10-4、沼×万2.6×10-4、嗜×深2.0×10-4、嗜×万2.3×10-4、深×万2.4×10-4.经影印法鉴定:形成的融合子可以分别生长于以相应的有机物为唯一碳源的培养基上,所有融合子体积均相当于两亲本株体积之和,融合子菌落形态特征介于两亲本株之间.从中随机挑选100个融合子,以辣椒苗作为靶标植物,从上述融合子中筛选到了1株具有显著促进作物生长、提高抗病性的融合子.     

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (＞1013 different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.     

FRIL蛋白体外维持脐血造血干/祖细胞的作用及细胞周期调控基因的表达

为探讨新的豆类凝集素(Flt3 receptor-interacting lectin,FRIL)体外维持脐血CD34^ 细胞的作用以及维持过程中细胞周期调控基因HTm4及HTm4S mRNA的表达及意义,我们利用FRIL维持培养脐血CD34^ 细胞,对其增殖曲线、细胞周期及集落形成能力进行常规分析,并用半定量RT—PCR法分别测定FRIL体外维持不同时间后脐血CD34^ 细胞中周期调控基因HTm4及HTm4S mRNA的表达变化。结果显示,FRIL培养的CD34^ 造血干／祖细胞的增殖趋势平缓,整个培养期间细胞增殖倍数不超过起始的3倍：14d之前,FRIL培养细胞的高增殖潜能集落形成细胞(HPP—CFC)形成集落数与FL组无差别,其后则维持高于FL的情况。细胞周期分析则显示,在28d的培养过程内,利用FRIL培养的细胞始终有80％以上维持在G0期;而周期调控基因HTm4及HTm4S在刚分离的脐血CD34^ 细胞中的表达水平较高;但培养1d后,几乎检测不到HTm4基因的表达;培养3～14d,该基因的表达回升并持续维持在高水平。而HTm4S基因的表达在第7d达最高水平,其余时间基本呈稳定表达。转染HTm4和HTm4S,亚细胞定位结果显示HTm4主要定位于核周围,而HTm4S则定位于整个胞浆,由此可能导致它们功能的区别。以上结果提示,长期培养体现出FRIL在维持造血干／祖细胞多能性上的优势;细胞周期调控基因HTm4及其新剪接子参与了FRIL体外长期维持脐血造血干／祖细胞处于静息状态的过程。     

间断低氧对大鼠下丘脑超微结构及前增食欲素水平的影响

目的探讨睡眠中间断低氧对大鼠下丘脑前增食欲素及受体水平的影响以及下丘脑超微结构的变化。方法大鼠分成对照组、间断低氧组和持续低氧组,分别给予吸入空气,持续低氧和间断低氧气体,并在实验开始后1d、3d、1w和4w应用RT-PCR方法测定大鼠下丘脑前增食欲素及受体水平,分析其间的变化关系,电镜观察下丘脑的超微结构变化。结果与对照组和持续低氧组比较,间断低氧4w后大鼠下丘脑前增食欲素mRNA水平明显降低,受体水平升高,但在持续低氧和对照组之间无明显差异。在低氧后1d、3d、7d后大鼠下丘脑前增食欲素mRNA降低,受体水平升高,在4w后,持续低氧组则接近正常。急性持续低氧大鼠超微结构变化更严重,而慢性间断低氧变化更持久。结论慢性间断低氧可以引起下丘脑前增食欲素下降及受体水平升高,急性持续低氧也可引起上述变化,而慢性持续低氧未引起增食欲素改变;慢性间断低氧大鼠下丘脑超微结构表现为严重而持久的变化。     

Inhibition of IFN-gamma receptor signaling by hepatitis C virus non-structural protein NS4B]

The function of NS4B is incompletely understood. The aim of the study is to understand the influence of NS4B on anti-viral response. After cell line stably expressing NS4B established, the influence of IFN-alpha of different concentration on VSV was studied using plaque assay; cell expression profiling caused by NS4B was studied using DNA microarray, and the IFNGR1 fluorescence intensity was analyzed. Our data showed that HCV-NS4B could suppress immuno-associated gene expression, in particular, IFN-gamma receptor signal transduction-related genes. Taken together, NS4B could play some roles in HCV resistance to IFN therapy.     

Diallyl trisulfide induces apoptosis and inhibits proliferation of A549 cells in vitro and in vivo

Lung cancer is the leading cause of cancer-related mortality all over the world. In recent years, pulmonary adenocarcinoma has surpassed squamous cell carcinoma in frequency and is the predominant form of lung cancer in many countries. Epidemiological investigations have shown an inverse relationship between garlic (Allium sativum) consumption and death rate from many cancers. Diallyl trisulfide (DATS) is one of the garlic-derived compounds (also known as: organosulfer compounds, OSC). DATS can induce apoptosis and inhibit the growth of many cancer cell lines. Our study demonstrated that the apoptotic incidents induced by DATS were a mitochondria-dependent caspase cascade through a significant decrease of the anti-apoptotic Bcl-2 that resulted in up-regulation of the ratio of Bax/Bcl-2 and the activity of caspase-3, -8, and -9. Eventually, DATS induced the apoptosis and inhibited the proliferation in a concentration- and time-dependent manner. Furthermore, by establishing an animal model of female BALB/c nude mice with A549 xenografts, we found that oral gavage of DATS significantly retarded growth of A549 xenografts in nude mice without causing weight loss or any other side effects compared with the control group. All the evidence both in vitro and in vivo suggested that DATS could be an ideal anti-cancer drug.