阴道毛滴虫铁氧还蛋白基因的原核表达

目的在原核细胞中表达阴道毛滴虫铁氧还蛋白(ferredoxin,Fd)基因。方法构建阴道毛滴虫Fd基因的原核表达重组质粒pUC19-Fd,转化大肠埃希菌JM109感受态细胞中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白质表达。结果经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western blot)分析,重组质粒在大肠埃希菌中表达出Fd。结论在大肠埃希菌中表达出了Fd。     

阴道毛滴虫多克隆抗体的制备及Fd基因的原核表达

目的 将阴道毛滴虫铁氧还蛋白（ferredoxin,Fd）基因在大肠埃希菌中诱导表达。方法制备阴道毛滴虫可溶性抗原,多点注射免疫家兔,获得的血清用ELISA测定其抗体效价。将原核表达重组质粒pET3C-Fd转化入大肠埃希菌BL21（DE3）感受态细胞中,异丙基-β-D-硫代半乳糖苷（IPTG）诱导蛋白质表达。结果制备出抗阴道毛滴虫多克隆抗体,抗体效价在1：8000以上,用于免疫印迹实验。经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹（Western blot）分析,重组质粒在大肠埃希菌中表达出Fd。结论在大肠埃希菌中表达出了Fd。     

紫色非硫光合细菌Rhodopseudomonas capsulata铁氧还蛋白(Ferredoxin)一些特性的研究

作者报导了(朱长喜等1980)从紫色非硫光合细菌Rhodopseudomonas capsulata N-3菌株中,分离纯化获得了聚丙烯酰胺凝胶电泳纯的铁氧还蛋白(Ferredoxin),通过对其生物活性、分子量的测定,铁、硫含量的化学分析以及电子自旋共振波谱(EPR)的研究,确认它是具有8Fe—8S簇活性中心结构的铁—硫蛋白。已知铁氧还蛋白的生物化学特性随它的来源有变化,因而表现它作为电子传递载体,在光合作用、生物固氮、氢代谢等方面的电子传递过程中,行使的功能也有差异(Rao和Hall 1977),为了进一步研究Rps.capsulata铁氧还蛋白的生理功能,我们测定了它的氨基酸组份,克分子消光系数以及与细菌载色体光合膜的结合状态等生化特性。     

荞麦黄酮复合物体外抗滴虫试验研究

采用试管二倍稀释法观察荞麦黄酮复合物对阴道毛滴虫体外抑制或杀伤作用,并以甲硝唑和洁尔阴为对照,测定受试药物对滴虫的最低致死浓度(MLC)和抑制率。结果显示:荞麦黄酮复合物较高浓度时对阴道滴虫有杀灭作用,其MLC为:33.75mg/ml生药(稀释度1:8)。其对阴道滴虫的杀伤作用与洁尔阴相似,但弱于甲硝唑。表明荞麦黄酮复合物高浓度(1:8)对阴道滴虫有良好的杀灭作用。     

乳酸杆菌的代谢产物对体外培养的阴道毛滴虫的影响

目的研究乳酸杆菌代谢产物对体外培养的阴道毛滴虫的杀伤作用。方法检测不同浓度的乳酸杆菌代谢产物对体外培养的阴道毛滴虫在不同作用时间下的杀伤率。结果乳酸杆菌代谢产物浓度10%,作用时间2 h、4 h和6 h体外培养毛滴虫的杀伤率分别为26.43%、37.47%和46.35%;浓度25%时杀伤率分别为43.56%、74.65%和90.15%;浓度50%杀伤率分别为92.36%、95.23%和99.01%。结论乳酸杆菌代谢产物的浓度越高,对体外培养的毛滴虫的杀伤力越大,作用时间越长,效果越好。     

蒙药金莲花体外抗阴道毛滴虫效果观察

目的观察金莲花对体外阴道毛滴虫的杀灭效果。方法采用不同浓度的金莲花水提物进行体外抗阴道毛滴虫试验,于药物作用后不同时间记录阴道毛滴虫的死亡率。结果金莲花具有抑制和杀灭阴道毛滴虫的作用,最低有效浓度为20.00 mg/mL。结论金莲花对阴道毛滴虫具有杀灭效果。     

NADPH依赖性硫氧还蛋白还原酶C在植物质体中的作用

硫氧还蛋白的氧化还原调节作用在生物界中普遍存在。它能够还原目标蛋白的二硫键,而自身的活性位点则被氧化。因此,对于新的催化循环,则需要由相应的还原酶将其再次还原成活性形式。硫氧还蛋白对维持高等植物的光合效率同样具有重要意义。叶绿体中的硫氧还蛋白分别由铁氧还蛋白依赖性硫氧还蛋白还原酶和NADPH依赖性硫氧还蛋白还原酶C(NTRC)两种酶还原。NTRC的本质是一种黄素蛋白,除了具有还原酶活性外,还整合了一个硫氧还蛋白结构域,在叶绿体和淀粉体的氧化还原调节中处于核心地位。这种特殊的双功能酶在卡尔文-本森循环、氧化戊糖磷酸途径、抗过氧化、四吡咯代谢、ATP和淀粉合成、生长素和光周期调控中扮演了多重角色。本综述总结了NTRC的生理功能,并讨论了该蛋白质对植物质体氧化还原稳态的调节机制。     

利用微型计算机分子图形技术显示铁氧还蛋白的分子结构

我们利用微型计算机图形技术对两种铁氧还蛋白的分子结构进行了研究,输入蛋白晶体数据,重构出铁氧还蛋白分子的三维结构模型,并可使之统X轴、Y轴和Z轴旋转;计算出肽链上原子之间的健长、健角、(?)角:显示微区结构;进一步可使两个铁氧还蛋白的三维结构图叠合,(?)利比较分析.实验表明,微型计算机分子图开技术在研究蛋白质等生物大分子结构,运动及中与功能的关系方面是有效的工具;     

探讨荆芥体外抗阴道毛滴虫的效果

目的探讨荆芥体外抗阴道毛滴虫的效果。方法将不同浓度的荆芥水提液作用于体外培养的阴道毛滴虫,于药物作用后不同时问记录毛滴虫的死亡率,并在光镜下观察药物作用前后毛滴虫的形态变化,同时与白头翁和青蒿的体外杀虫效果相比较。结果荆芥的杀虫效果与药物浓度和作用时间呈正相关。荆芥水提液触杀阴道毛滴虫的最低有效浓度为1：4。3种中药中,白头翁的杀虫效果最好,与另2种中药相比较差异有非常显著性（P〈0．01）;荆芥与青蒿的作用效果接近（P〉0．05）。荆芥作用后毛滴虫体内充满大量颗粒和空泡,部分虫体裂解、内容物外溢。结论荆芥具有较强的抗阴道毛滴虫作用。     

荆芥体外抗阴道毛滴虫作用机制的实验研究

目的探讨荆芥体外杀灭阴道毛滴虫的作用机制。方法将浓度为1∶4的荆芥水提液作用于体外培养的阴道毛滴虫,并采用透射电镜观察经药物作用2 h和4 h后阴道毛滴虫的超微结构变化。结果荆芥作用后阴道毛滴虫多聚核糖体解聚,粗面内质网脱颗粒,胞质内可见大量空泡;随药物作用时间延长,核膜不完整,核质变疏松;最终,胞膜破损,内容物外溢,虫体死亡。结论荆芥可破坏阴道毛滴虫的内膜系统,具有较强的抗滴虫作用。     

Keto-acid oxidoreductases in the anaerobic protozoa.

In anaerobes, decarboxylation of pyruvate is executed by the enzyme pyruvate:ferredoxin oxidoreductase, which donates electrons to ferredoxin. The pyruvate:ferredoxin oxidoreductase and its homologues utilise many alternative substrates in bacterial anaerobes. The pyruvate:ferredoxin oxidoreductase from anaerobic protozoa, such as Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica have retained this diversity in usage of alternative keto acids for energy production utilising a wide variety of substrates. In addition to this flexibility, both T. vaginalis and G. duodenalis have alternative enzymes that are active in metronidazole-resistant parasites and that do not necessarily involve donation of electrons to characterized ferredoxins. Giardia duodenalis has two oxoacid oxidoreductases, including pyruvate:ferredoxin oxidoreductase and T. vaginalis has at least three. These alternative oxoacid oxidoreductases apparently do not share homology with the characterized pyruvate:ferredoxin oxidoreductase in either organism. Independently, both G. duodenalis and T. vaginalis have retained alternative oxoacid oxidoreductase activities that are clearly important for the survival of these parasitic protists.     

Trichomonads, hydrogenosomes and drug resistance

Trichomonas vaginalis and Tritrichomonas foetus are sexually transmitted pathogens of the genito-urinary tract of humans and cattle, respectively. These organisms are amitochondrial anaerobes possessing hydrogenosomes, double membrane-bound organelles involved in catabolic processes extending glycolysis. The oxidative decarboxylation of pyruvate in hydrogenosomes is coupled to ATP synthesis and linked to ferredoxin-mediated electron transport. This pathway is responsible for metabolic activation of 5-nitroimidazole drugs, such as metronidazole, used in chemotherapy of trichomoniasis. Prolonged cultivation of trichomonads under sublethal pressure of metronidazole results in development of drug resistance. In both pathogenic species the resistance develops in a multistep process involving a sequence of stages that differ in drug susceptibility and metabolic activities. Aerobic resistance, similar to that occurring in clinical isolates of T. vaginalis from treatment-refractory patients, appears as the earliest stage. The terminal stage is characterised by stable anaerobic resistance at which the parasites show very high levels of minimal lethal concentration for metronidazole under anaerobic conditions (approximately 1000 microg ml(-1)). The key event in the development of resistance is progressive decrease and eventual loss of the pyruvate:ferredoxin oxidoreductase so that the drug-activating process is averted. In T. vaginalis at least, the development of resistance is also accompanied by decreased expression of ferredoxin. The pyruvate:ferredoxin oxidoreductase deficiency completely precludes metronidazole activation in T. foetus, while T. vaginalis possesses an additional drug-activating system which must be eliminated before the full resistance is acquired. This alternative pathway involves the hydrogenosomal malic enzyme and NAD:ferredoxin oxidoreductase. Metronidazole-resistant trichomonads compensate for the hydrogenosomal deficiency by an increased rate of glycolysis and by changes in their cytosolic pathways. Trichomonas vaginalis enhances lactate fermentation while T. foetus activates pyruvate conversion to ethanol. Drug-resistant T. foetus also increases activity of the cytosolic NADP-dependent malic enzyme, to enhance the pyruvate producing bypass and provide NADPH required by alcohol dehydrogenase. Production of succinate by this species is abolished. Metabolic changes accompanying in-vitro development of metronidazole resistance demonstrate the versatility of trichomonad metabolism and provide an interesting example of how unicellular eukaryotes can adjust their metabolism in response to the pressure of an unfavorable environment.     

Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance

Ferredoxin, Fd, is often deficient in metronidazole-resistant strains of Trichomonas vaginalis and is thought to be necessary for drug activation. To directly test whether Fd is essential for metronidazole susceptibility, gene replacement technology has been developed for T. vaginalis. The selectable marker gene neomycin phosphotransferase (NEO) flanked by approximately 2.6 and approximately 2.0 kBp of the Fd 5' and 3' flanking regions (pKO-FD-NEO) was introduced into cells on linear DNA and selected for NEO gene expression. Stable transformants were shown to contain the NEO gene in the Fd locus and to have completely lost the Fd gene. Northern and immunoblot analyses confirm the loss of Fd mRNA and protein in pKO-FD-NEO cells. Analyses of the activity of hydrogenosomal proteins in Fd KO cells show a fourfold increase in hydrogenase activity and a 95% decrease in pyruvate/ferredoxin oxidoreductase (PFO) activity. In contrast, PFO and hydrogenase mRNA levels are unchanged. Surprisingly, Fd KO cells are not resistant to metronidazole under aerobic or anaerobic conditions. These cells are capable of producing molecular hydrogen, albeit at 50% the level of the parental strain, demonstrating that the Fd gene product eliminated in KO cells is neither necessary for hydrogen production nor metronidazole activation. Together these data indicate the presence of unidentified Fds or flavodoxins capable of drug activation or an unidentified mechanism that does not require either PFO or Fd for metronidazole activation.     

Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance

Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica , Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we report that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thioredoxin reductase was greatly diminished in extracts of metronidazole-treated cells and intracellular non-protein thiol levels were sharply decreased. We generated a highly metronidazole-resistant cell line that displayed only minimal thioredoxin reductase activity, not due to diminished expression of the enzyme but due to the lack of its FAD cofactor. Reduction of free flavins, readily observed in metronidazole-susceptible cells, was also absent in the resistant cells. On the other hand, iron-depleted T. vaginalis cells, expressing only minimal amounts of PFOR and hydrogenosomal malate dehydrogenase, remained fully susceptible to metronidazole. Thus, taken together, our data suggest a flavin-based mechanism of metronidazole activation and thereby challenge the current model of hydrogenosomal activation of nitroimidazole drugs.     

Mycoplasma hominis in Cuban Trichomonas vaginalis isolates: association with parasite genetic polymorphism

Trichomonas vaginalis can be naturally infected with intracellular Mycoplasma hominis. This bacterial infection may have implications for trichomonal virulence and disease pathogenesis. The objective of the study was to report the presence of M. hominis in Cuban T. vaginalis isolates and to describe the association between the phenotype M. hominis infected with RAPD genetic polymorphism of T. vaginalis. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 40 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with M. hominis. The trees drawn based on RAPD data showed no relations with metronidazole susceptibility and significantly association with the presence of M. hominis (P=0.043), which demonstrates the existence of concordance between the genetic relatedness and the presence of M. hominis in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the bacterial enters and/or survival.     

Trichomonas species: homocysteine desulphurase and serine sulphydrase activities

Homocysteine desulphurase (EC 4.4.1.2) and serine sulphydrase (EC 4.2.1.22) activities in various lines of Trichomonas vaginalis, both metronidazole resistant and sensitive, and other trichomonad species were assessed. T. vaginalis contained the highest homocysteine desulphurase and serine sulphydrase activities of all the species. Although the levels of the enzyme activity in T. vaginalis isolates differed, no correlation between the activities and sensitivity to metronidazole was apparent. T. vaginalis homocysteine desulphurase catalysed both the hydrolysis of homocysteine to hydrogen sulphide, ammonia, and 2-oxoacid, and an exchange reaction between homocysteine and 2-mercaptoethanol. Homocysteine desulphurase was detected as a single enzyme band on isoelectric focusing, whereas several isoenzymes of serine sulphydrase were found. There were large differences in serine sulphydrase isoenzyme patterns between T. vaginalis lines and between species. Several isoenzymes were amplified in cells grown with 10(-5) M DL-propargylglycine for 24 hr. T. vaginalis homocysteine desulphurase and serine sulphydrase activities were inhibited by bithionol, hexachlorophene, and dichlorophene. These compounds also inhibited growth in vitro of T. vaginalis at concentrations similar to those that inhibited the enzymes.     

Methyl jasmonate induces cell cycle block and cell death in the amitochondriate parasite Trichomonas vaginalis

Jasmonates are a group of small lipids produced in plants and function as stress hormones. They are selectively cytotoxic against cancer cells. Methyl jasmonate (MJ), one of the naturally occurring jasmonates, has direct mitochondriotoxic effects, strongly suggesting that mitochondria are target organelles of jasmonates. We have previously shown that jasmonates are cytotoxic to two human parasites -Schistosoma mansoni and Plasmodium falciparum. Both the cancer cells and the parasites mentioned above possess mitochondria. The present work aimed to examine whether jasmonates are able to damage cells lacking mitochondria, e.g., some unicellular human parasitic flagellates. We found that MJ induced death of the amitochondriate Trichomonas vaginalis parasites. MJ caused fragmentation and condensation of the DNA of T. vaginalis, resembling phenomena associated with apoptotic death. However, DNA laddering, a sub-G(1) cell cycle stage peak and caspase-3 activation were not observed. Thus, MJ-induced T. vaginalis cell death appears to be non-apoptotic. We found that MJ induced cell cycle block at the G(2)/M phase in T. vaginalis, similar to the effect of metronidazole. We examined the influence of MJ on the bioenergetic pathways of T. vaginalis, and found that depletion of ATP did not precede death of the parasites, but rather reflected it. Nevertheless, 2-deoxy-d-glucose, a glycolysis blocker, was synergistic with MJ in causing death of T. vaginalis cells, suggesting that MJ does perturb the bioenergetic homeostasis of the parasites. Finally, MJ was found to be cytotoxic towards a metronidazole-resistant strain of T. vaginalis (ATCC 50143), suggesting that it may be effective for the treatment of nitroimidazole-refractory trichomoniasis. In conclusion, MJ was found to exhibit mitochondria-independent cytotoxicity and presents a potentially novel agent against T. vaginalis.     

An Increase in Helicobacter pylori Strains Resistant to Metronidazole: A Five-Year Study

Background. Metronidazole is one of the most commonly used antimicrobial agents for the treatment of Helicobacter pylori infection. Resistance to metronidazole has been reported worldwide but with a wide range of prevalence. We started using the classical triple therapy (bismuth, tetracycline, and metronidazole) for H. pylori infection in 1991 but recently have experienced a decline in its efficacy in curing the infection. Thus our aim was to investigate in a single center the prevalence of metronidazole-resistant H. pylori over a period of 5 years.
Materials and Methods. A total of 1,015 different H. pylori strains collected over a period of 5 years were tested for sensitivity against metronidazole, ampicillin, tetracycline, and imipenem. Antibiotic sensitivity was tested by the disk diffusion and agar dilution methods. To elucidate further the possible relationship between these metronidazole-resistant strains, genomic DNA digestion by the Hae III endonuclease and ribotyping were undertaken in a selected group of isolates.
Results. In 1991, 29 of 132 (22.0%) tested strains of H. pylori were found to be resistant to metronidazole. Since our initiation at that time of a triple therapy of bismuth, metronidazole, and tetracycline, the prevalence of metronidazole-resistant strains rose rapidly to 73.2% in 1995. All H. pylori isolates were sensitive to ampicillin, tetracycline, and imipenem. A high degree of genomic heterogeneity was found among these isolates. Thus it is unlikely that the resistant strains of H. pylori were originated from a single clone.
Conclusions. This study shows a rapid increase in metronidazole-resistant H. pylori with the use of an anti- Helicobacter regimen that contains metronidazole. We anticipate that the efficacy of metronidazole-containing anti- Helicobacter regimens will decline with the rapid rise in resistant strains of H. pylori.     

The crystal structure of Trichomonas vaginalis ferredoxin provides insight into metronidazole activation

Crystallographic studies revealing the three-dimensional structure of the oxidized form of the [2Fe-2S] ferredoxin from Trichomonas vaginalis (TvFd) are presented. TvFd, a member of the hydrogenosomal class of ferredoxins, possesses a unique combination of redox and spectroscopic properties, and is believed to be the biological molecule that activates the drug metronidazole reductively in the treatment of trichomoniasis. It is the first hydrogenosomal ferredoxin to have its structure determined. The structure of TvFd reveals a monomeric, 93 residue protein with a fold similar to that of other known [2Fe-2S] ferredoxins. It contains nine hydrogen bonds to the sulfur atoms of the cluster, which is more than the number predicted on the basis of the spectroscopic data. The TvFd structure contains a large dipole moment like adrenodoxin, and appears to have a similar interaction domain. Our analysis demonstrates that TvFd has a unique cavity near the iron-sulfur cluster that exposes one of the inorganic sulfur atoms of the cluster to solvent. This cavity is not seen in any other [2Fe-2S] ferredoxin with known structure, and is hypothesized to be responsible for the high rate of metronidazole reduction by TvFd.     

Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis

Trichomoniasis is the most common, sexually transmitted infection.It is caused by the flagellated protozoan parasite Trichomonas vagina/is. Symptoms include vaginitis and infections have been associatedwith preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDSand cervical cancer. Trichomoniasis has the highest prevalence and incidence of any sexually transmitted infection. The 5-nitroimidazole drugs, of which metronidazole is the most prescribed, are the only approved,effective drugs to treat trichomoniasis. Resistance against metronidazole is frequently reported and cross-resistance among the family of 5-nitroimidazole drugs is common, leaving no alternative for treatment, withsome cases remaining unresolved. The mechanism of metronidazole resistance in T. Vagina/is from treatment failures is not well understood, unlike resistance which is developed in the laboratory under increasingmet ronidazole pressure. In the latter situation, hydrog enosomal function which is involved in activationof the prodrug, metronidazole, is down-regulated. Reversion to sensitivity is incomplete after removal ofdrug pressure in the highly resistant parasites while clinically resistant strains, so far analysed, maintaintheir resistance levels in the absence of drug pressure. Although anaerobic resistance has been regarded asa laboratory induced phenomenon, it clearly has been demonstrated in clinical isolates. Pursuit of both approaches will allow dissection of the underlying mechanisms. Many alternative drugs and treatments have been tested in vivo in cases of refractory trichomoniasis, as well as in vitro with some successes including the broad spectrum anti-parasitic drug nitazoxanide. Drug resistance incidence in T. Vagina/is appears to be on the increase and improved surveillance of treatment failures is urged.