Colicin M is only bactericidal when provided from outside the cell

Summary The colicin M structural gene, cma, was subcloned in a vector which allowed temperature-inducible control of its expression. Induction of expression of cma in colicin M uptake proficient strains was lethal for the host cell when the colicin M immunity protein was not present. In liquid culture cells lysed, and no colonies were formed on solid media. These effects were not observed in mutants defective in the colicin receptor (FhuA) or uptake functions (TonB, TolM), nor in wild-type cells treated with trypsin prior to induction of cma expression. It was concluded that cytoplasmic colicin M is not toxic for the producing cell. To exert a lethal effect the colicin has to enter the cell from outside. Cells expressing cma released small amounts of colicin M.     

Anti-Helicobacter pylori activity of Plumbago zeylanica L

It has been shown that the presence of infection by Helicobacter pylori is strongly associated with gastric cancer and peptic ulceration. In western medicine, a 3-fold therapeutic regimen, emphasizing the use of antibiotics, is typically used to suppress H. pylori activity. However, antibiotic drug resistance frequently develops as a consequence of such treatment. In our previous study, 50 Taiwanese folk medicinal plants were screened for their anti-H. pylori activities. The results revealed that Plumbago zeylanica L. had the highest inhibitory effects against H. pylori. In this study, therefore, we have focused on establishing the anti-H. pylori activities of P. zeylanica L. Water and the organic solvents ethanol, ethyl acetate and acetone were used for P. zeylanica L. extraction, obtaining yields of 1.66-6.84% (w/w). Excluding the water extract, higher anti-H. pylori activity was demonstrated for all the extracts, both using the agar diffusion and dilution methods. The ethyl acetate extract exhibited the lowest minimum inhibitory concentrations against five H. pylori strains, of which ranged from 0.32 to 1.28 mg ml-1, followed, in ascending order, by the acetone, ethanol and water analogs. Bactericidal activity was determined for P. zeylanica L. extracts, with the lowest minimum bactericidal concentrations (5.12-20.48 mg ml-1) demonstrated for the ethyl acetate, followed, in ascending order, by the acetone and ethanol analogs. Bactericidal activity appeared to be in a dose-dependent manner. Through a broad pH range (2-7), bactericidal activity was not affected when extract concentrations were greater than or equal to the minimum bactericidal concentration. High stability was demonstrated for the ethyl acetate P. zeylanica L. extract within pH range of 1-7, exhibiting all pH treatments bactericidal activity.     

Synergy of aminoglycoside antibiotics by 3-Benzylchroman derivatives from the Chinese drug Caesalpinia sappan against clinical methicillin-resistant Staphylococcus aureus (MRSA)

The in vitro antimicrobial activities of three 3-Benzylchroman derivatives, i.e. Brazilin (1), Brazilein (2) and Sappanone B (3) from Caesalpinia sappan L. (Leguminosae) were assayed, which mainly dealt with synergistic evaluation of aminoglycoside and other type of antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) by the three compounds through the Chequerboard and Time-kill curve methods. The results showed that Compounds 1–3 alone exhibited moderate to weak activity against methicillin-susceptible S. aureus (MSSA) and other standard strains by MICs/MBCs ranged from 32/64 to >1024/>1024 μg/ml, with the order of activity as 1 > 2 > 3. Chequerboard method showed significant anti-MRSA synergy of 1/Aminoglycosides (Gentamicin, Amikacin, Etimicin and Streptomycin) combinations with (FICIs)₅₀ at 0.375–0.5. The combined (MICs)₅₀ values (μg/ml) reduced from 32–128/16–64 to 4–8/4–16, respectively. The percent of reduction by MICs ranged from 50% to 87.5%, with a maximum of 93.8% (1/16 of the alone MIC). Combinations of 2 and 3 with Aminoglycosides and the other antibiotics showed less potency of synergy. The dynamic Time-killing experiment further demonstrated that the combinations of 1/aminoglycoside were synergistically bactericidal against MRSA. The anti-MRSA synergy results of the bacteriostatic (Chequerboard method) and bactericidal (time-kill method) efficiencies of 1/Aminoglycoside combinations was in good consistency, which made the resistance reversed by CLSI guidelines. We concluded that the 3-Benzylchroman derivative Brazilin (1) showed in vitro synergy of bactericidal activities against MRSA when combined with Aminoglycosides, which might be beneficial for combinatory therapy of MRSA infection.     

Synthesis and biological activity of [L-3,4-dehydroproline3]-tuftsin

A 3,4-dehydroproline analogue of tuftsin (L-Thr-L-Lys-L-Pro-L-Arg) was prepared by the solid phase synthetic method. Following reversed-phase high performance liquid chromatography (HPLC) purification, the analogue was compared to tuftsin for its ability to enhance the chemotactic, bactericidal and phagocytic activities of polymorphonuclear leukocytes (PMN). Both tuftsin and [Δ³-pro³]-tuftsin elicited a similar significant chemotactic effect at a concentration of 10 μg/ml. A slight suppression of the chemotactic activity was observed with tuftsin at 10^?3 μg/ml and with [Δ³-pro³]-tuftsin at concentrations of 10^?3, 10^?2 (significant) and 10^?1 μg/ml. Although similar bactericidal activities were observed for both peptides, PMN exposed to [Δ³-pro³]-tuftsin exhibited increased phagocytic indicies 2–4 times that of tuftsin-treated PMN at concentrations of 0.4, 0.6 and 1.0 μg/ml.     

利用GAP启动子在毕赤酵母中组成型表达人鹅型溶菌酶2

利用甘油醛三磷酸脱氢酶(glyceraldehydes-3-phosphatedehydrogenase,GAP)启动子在毕赤酵母中表达人鹅型溶菌酶2(human goose-type lysozyme 2,h LysG2),并在小试规模建立一套有效的重组hLysG2(recombinant h LysG2,rh LysG2)生产工艺流程。根据毕赤酵母密码子偏爱性设计并人工合成hLysG2基因,将其连接至pGAPZαA质粒中,构建重组表达质粒pGAPZαA-h LysG2。将重组表达载体线性化后电转化毕赤酵母GS115感受态细胞,通过Zeocin抗性筛选获取高拷贝重组菌株,并在5L生物反应器中进行发酵培养。发酵60h后发酵液上清酶活性达到最高,发酵液上清经SDS-PAGE及Western blot检测证实rh LysG2得到表达。与诱导型表达相比,组成型表达发酵时间缩短了48h,上清中rhLysG2总活性提高了23.8%;使用甲壳素亲和层析和分子筛层析对rhLysG2进行纯化后,每升发酵液上清可纯化到187.4mg重组蛋白,纯化产物纯度达99.0%以上;浊度测定法分析显示,在p H 5.6、30℃和0.1mol/L Na+的条件下,rhLysG2可达到最大酶活性13 500U/mg。利用GAP启动子在毕赤酵母中成功表达了高纯度和高活性的rh LysG2,避免了甲醇的使用,缩短了发酵时间,提高了蛋白产量,为将rhLysG2开发为新型抗耐药菌药物奠定了基础。     

Bactericidal effect of Naja nigricollis toxin γ is related to its membrane-damaging activity

The aim of the present study is to investigate the causal relationship between membrane-damaging activity and bactericidal activity of Naja nigricollis toxin γ. Toxin γ showed a similar inhibitory activity on the growth of Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria). Antibacterial activity of toxin γ correlated positively with increase in membrane permeability of bacterial cells. Morphological examination showed that toxin γ disrupted the integrity of bacterial membrane. Toxin γ showed similar binding capability with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and destabilization of LPS layer and inhibition of LTA biosynthesis on cell wall increased bactericidal effect of toxin γ on E. coli and S. aureus, respectively. Although the potency of toxin γ on permeabilzing model membrane of E. coli and S. aureus was similar, the mode of interaction between toxin γ and model membrane of E. coli and S. aureus differed. Membrane-damaging activity of toxin γ was inhibited by either LPS or LTA. Nevertheless, LPS and LTA altered differently membrane-bound conformation of toxin γ. Taken together, our data suggest that bactericidal activity of toxin γ depends on its ability to induce membrane permeability, and that LPS and LTA structurally suppresses bactericidal effect of toxin γ.     

Lethal toxin of Bacillus anthracis causes apoptosis of macrophages

Lethal toxin is a major anthrax virulence factor, causing the rapid death of experimental animals. Lethal toxin can enter most cell types, but only certain macrophages and cell lines are susceptible to toxin-mediated cytolysis. We have shown that in murine RAW 264.7 cells, sublytic amounts of lethal toxin trigger intracellular signaling events typical for apoptosis, including changes in membrane permeability, loss of mitochondrial membrane potential, and DNA fragmentation. The cells were protected from the toxin by specific inhibitors of caspase-1, -2, -3, -4, -6, and -8. Phagocytic activity of macrophages was inhibited by sublytic concentrations of lethal toxin. Infection of cells with anthrax (Sterne) spores impaired their bactericidal capacity, which could be reversed by a lethal toxin inhibitor, bestatin. We suggest that apoptosis rather than direct lysis is biologically relevant to lethal toxin intracellular activity.     

人源溶菌酶基因LYZL4在毕赤酵母中的重组表达及活性测定

LYZL4是本实验室克隆的一种c型人溶菌酶基因,根据毕赤酵母密码子偏爱性对LYZL4基因进行优化设计,优化后的基因克隆至具有乙醇氧化酶启动子(AOX1)的pPIC9K载体中。通过电转化方法整合到甲醇营养型毕赤酵母表达菌株GS115基因组上,再利用不同浓度梯度的G418抗性YPD固体培养基筛选高拷贝转化子,经摇瓶发酵后,其上清液在SDS-PAGE胶上14.4 kDa处出现明显的蛋白条带,该蛋白条带经质谱鉴定证实是人LYZL4蛋白。以人溶菌酶(164 071U/mg)作为标准品,使用艳红微球菌作为底物,通过比色法测定重组LYZL4溶菌酶蛋白的活性,结果显示重组人溶菌酶LYZL4蛋白具有明显的溶菌活性,其酶活力可达38893U/ml。     

A novel method for preparation of HAMLET-like protein complexes

Some natural proteins induce tumor-selective apoptosis. α-Lactalbumin (α-LA), a milk calcium-binding protein, is converted into an antitumor form, called HAMLET/BAMLET, via partial unfolding and association with oleic acid (OA). Besides triggering multiple cell death mechanisms in tumor cells, HAMLET exhibits bactericidal activity against Streptococcus pneumoniae. The existing methods for preparation of active complexes of α-LA with OA employ neutral pH solutions, which greatly limit water solubility of OA. Therefore these methods suffer from low scalability and/or heterogeneity of the resulting α-LA - OA samples. In this study we present a novel method for preparation of α-LA - OA complexes using alkaline conditions that favor aqueous solubility of OA. The unbound OA is removed by precipitation under acidic conditions. The resulting sample, bLA-OA-45, bears 11 OA molecules and exhibits physico-chemical properties similar to those of BAMLET. Cytotoxic activities of bLA-OA-45 against human epidermoid larynx carcinoma and S. pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae with bLA-OA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are markedly rescued from death upon pretreatment with an inhibitor of Ca²⁺ transport. Hence, the activation mechanisms of S. pneumoniae death are analogous for these two complexes. The developed express method for preparation of active α-LA - OA complex is high-throughput and suited for development of other protein complexes with low-molecular-weight amphiphilic substances possessing valuable cytotoxic properties.     

Effect of methanol and Me2SO exposure on mitochondrial activity and distribution in stage III ovarian follicles of zebrafish (Danio rerio)

In this study the effect of cryoprotectants that have been shown to be the least toxic to zebrafish ovarian follicles (methanol and Me₂SO), on mitochondria of stage III ovarian follicles was evaluated. The mitochondrial distributional arrangement, mitochondrial membrane potential, mtDNA copy number, ATP levels and ADP/ATP ratios were assessed following exposure to cryoprotectants for 30 min at room temperature. Results obtained by confocal microscopy showed that 30 min exposure to 2 M methanol induced a loss of membrane potential, although viability tests showed no decrease in survival even after 5 h post-exposure incubation. Higher concentrations of methanol (3 and 4 M) induced not only a decrease in mitochondrial membrane potential but also the loss of mitochondrial distributional arrangement, which suggested a compromised mitochondrial function. Furthermore 3 and 4 M treatments resulted in a decrease in viability assessed by Fluorescein diacetate–Propidium iodide (FDA–PI) and in a decrease in mtDNA copy number and ADP/ATP ratio after 5 h incubation following methanol exposure, indicating a delayed effect. The use of Me₂SO, which is considered to be a more toxic CPA to zebrafish ovarian follicles than methanol, caused a decrease in viability and a sustained decrease in ATP levels accompanied by failure to maintain mtDNA copy number within 1 h post-exposure incubation. These results indicated that even CPAs that are considered to have no toxicity as determined by Trypan blue (TB) and FDA–PI tests can have a deleterious effect on mitochondrial activity, potentially compromising oocyte growth and embryo development.     

经济杀菌灭藻剂OACL控制工业循环水中有害微生物的研究

研究了经济杀菌灭藻剂OACL对异氧菌、硫酸盐还原菌和铁细菌的杀菌作用。结果表明,在实验室条件下,其杀菌率平均为99％以上,在pH5和pH7时分别为97.8％和88％。而在现场投药试验条件下为92％以上。可以认为,OACL是一种新型的、经济的高效杀菌灭藻剂。