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.     

Cell cytotoxicity of sodium nitrite, sodium nitroprusside and Roussin's black salt against Trichomonas vaginalis

Abstract We have investigated the action of sodium nitrite and other nitrosyl complexes, such as sodium nitroprusside and Roussin's black salt, on the growth of metronidazole-sensitive and resistant strains of Trichomonas vaginalis and their hydrogenosomal enzymes. All three chemicals inhibited the growth of T. vaginalis : sodium nitrite at 8 mM, sodium nitroprusside at 1.2 mM and Roussin's black salt at 0.2 mM. Metronidazole-sensitive (KT9) and resistant (CDC85) isolates showed similar cytotoxicity against these molecules. Specific activities of pyruvate:ferredoxin oxidoreductase and hydrogenase and oxygen uptake rates were decreased in the T . vaginalis isolate treated with sodium nitrite and sodium nitroprusside. However, Roussin's black salt increased the specific activity of pyruvaterferredoxin oxidoreductase or hydrogenase in CDC85 or KT9 cells and increased the oxygen uptake rate in the KT9 isolate.     

Lysogenic phages of Closrtidium perfringens: mapping of the chromosomal attachment sites

The sites of insertion for two lysogenic bacteriophages have been mapped on the chromosome of Clostridium perfringens strain CPN50 using two techniques based on pulsed field gel electrophoresis. Phage phi 29 was mapped to the 1 Mb region of the 3.6 Mb genome, near nanH which encodes a potential virulence factor, while phi 59 was found to have inserted at 2.9 Mb.     

Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.     

Determination of genome size and restriction pattern polymorphism of Rickettsia prowazekii and Rickettsia typhi by pulsed field gel electrophoresis

Abstract Pulsed field gel electrophoresis (PFGE) of Sma I, Mlu I and Sal I digested DNA was used to estimate genome size and perform restriction fragment length polymorphism analysis for Rickettsia prowazekii and Rickettsia typhi . We concluded that the genome of R. prowazekii and R. typhi consisted of a single chromosomal DNA. The total length of DNA of R. prowazekii was 1,106±54 kb and of R. typhi was 1,133±44kb. It was possibleto differentiate two strains of R. prowazekii , Breinl and EVir, by PFGE analysis after Sal I digestion. Restriction fragment length polymorphism analysis did not reveal intraspecies differences between three human isolates and one Xenopsilla cheopis isolate of R. typhi .     

氧化葡萄糖酸杆菌SCB329基因组的大小与结构的研究

收集维生素C产生菌氧化葡萄糖酸杆菌GluconobacteroxydansSCB32 9的纯培养对数期的菌体 ,采用凝胶包埋法制备完整染色体 ,用稀有酶切位点的限制性内切酶和脉冲场电泳技术对SCB32 9的基因组进行了分析 ,SpeⅠ (5′ ACTAGT)酶切有 2 4个片段 ,其大小从 1 0kb到32 0kb,用XbaⅠ (5′ TCTAGA)酶切产生 40个片段 ,其大小从 4kb到 2 0 0kb,综合两种限制酶酶切片段长度的总和结果 ,SCB32 9基因组大小为 2 70 0kb,SCB32 9基因组由一条 2 50 0kb的染色体和一个 2 4 5kb的质粒组成。通过用脱氧核糖核酸酶Ⅰ和S1核酸酶处理其基因组后电泳证实SCB32 9的染色体和质粒的拓扑学结构均为环状     

Short protocol for pulsed field gel electrophoresis of a variety of Clostridia species

While pulsed field gel electrophoresis has become an important tool for genotyping of bacteria, one of its drawbacks is that standard methods are rather time-consuming. In order to overcome this problem, shortened procedures for DNA preparation have been developed for some bacterial species. The aim of this study was to examine if a short procedure used for pulsed field gel electrophoresis of Clostridium botulinum could be applied to other Clostridia species. For this, the protocol was modified and used to prepare the DNA of 34 strains of 25 different Clostridia species. In contrast to a standard procedure, which takes at least 5 days from DNA extraction to completion of the electrophoresis, this protocol yielded results within 2 days. In order to directly compare the results of the short protocol with those of the standard, long procedure, parallel DNA preparations were performed using both methods and the two DNA samples thus obtained per strain were then run on the same gel. Briefly, the procedure was as follows. After embedding the bacterial cells in agarose, the agarose blocks were incubated for 1 h in lysis solution containing lysozyme, mutanolysin, lysostaphin and RNase. This was followed by a 1-h proteinase K treatment. Then, slices were cut from the agarose blocks and washed for 15 min in TE buffer, these washes were repeated four times with fresh TE. After a 2-h restriction with SmaI, electrophoresis was carried out overnight.     

交变脉冲电场凝胶电泳与植物大分子DNA的制备

介绍了交变脉冲电场凝胶电泳的原理、方法及其在植物大分子ＤＮＡ制备方面的应用     

用脉冲电场凝胶电泳检测电离辐射所致哺乳动物细胞DNA双链断裂

 本文将反向交变电场和六角形电极电场这两种脉冲电场凝胶电泳技术应用于X线照射小鼠乳癌细胞SR-1所致DNA双链断裂的检测,在本实验条件下,用这种电泳都能检测到低至1.5Gy照射所产生的DNA双链断裂,并且用六角形电极电场电泳获得了DNA双链断裂程度与照射剂量之间的良好线性关系,此外,还用此方法观察了不同浓度自由基清除剂DMSO对X线照射SR-1细胞所致DNA双链断裂的保护作用,结果进一步证实本方法的可靠性。     

ADPRT抑制剂对不同辐射敏感性人肿瘤细胞株的作用比较

从细胞的克隆形成能力和细胞DNA双链断裂及修复几方面分析了两个人卵巢癌细胞株HOC8和A2780对电离辐射的敏感性并探讨了ADP-核糖基转移酶（ADPR）的特异性抑制剂3-氨基苯甲酰胺（3－AB）对二者的辐射增敏效应,结果表明A2780细胞的辐射敏感性大大高于HOC8细胞,其D0值分别为0．9和2．5Gy;γ射线所致两株细胞的初始DNA双链断裂水平没有显著差异,但A2780细胞对DNA双链断裂的修复能力比HOC8细胞低．3AB能降低受照细胞的克隆形成能力及细胞对双链断裂的修复能力,其中对HOC8细胞的作用更为明显．     

Epidemiological evaluation of Neisseria meningitidis serogroup B by pulsed-field gel electrophoresis

Abstract Genomic DNA from 25 strains of serogroup B Neisseria meningitidis was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with Spe I. N. meningitidis genomic DNA displayed considerable diversity. The diversity we observed among these strains was stable and included isolates from an outbreak that were phenotypically identical. This confirms the value of macrorestriction profiling and PFGE in providing epidemiologically stable strain markers for typing meningococci.     

Estimation of genome sizes of hyperthermophiles

Genomes of various hyperthermophilic and extremely thermophilic prokaryotes were analyzed with respect to size, physical organization, and 16S rDNA copy number. Our results show that all the genomes are circular, and they are in the size range of 1.6–1.8 Mb for Pyrodictium abyssi, Methanococcus igneus, Pyrobaculum aerophilum, Archaeoglobus fulgidus, Archaeoglobus lithotrophicus, and Archaeoglobus profundus (the two bacteria Fervidobacterium islandicum and Thermosipho africanus possess genomes of 1.5-Mb size). A systematic study of all validly described species of the order Sulfolobales revealed the existence of two classes of genome size for these archaea, correlating with phylogenetic analyses. The Metallosphaera–Acidianus group, plus Sulfolobus metallicus, have genomes of ca. 1.9 Mb; the other members of the order Sulfolobales group possess genomes >2.7 Mb. The special case of Stygiolobus azoricus is discussed. Received: August 10, 1997 / Accepted: January 1, 1998     

Asymmetric fusion between protoplasts of tomato (Lycopersicon esculentum Mill.) and gamma-irradiated protoplasts of potato (Solanum tuberosum L.): the effects of gamma irradiation

This paper describes the aggregation of nuclei in heterokaryons of tomato and unirradiated or irradiated potato protoplasts and the effects of gamma irradiation of potato and tomato protoplasts on single- and double-stranded DNA fragmentation, DNA repair and DNA synthesis as revealed by alkaline and pulsed field gel electrophoresis and an immunocytochemical technique. The prospects for obtaining highly asymmetric somatic hybrids of tomato and gamma-irradiated potato are discussed.     

从植物细胞核分离大分子量核DNA

研究了从植物中分离百万碱基对级大分子量核DNA的方法。该方法利用差速离心分离植物细胞核,经低熔点琼脂糖块或低熔点琼脂糖微珠包埋,蛋白酶K原位裂解后制备大分子量核DNA。结果表明,选择不同生长时期的材料和不同的包埋细胞核方式对大分子量核DNA的制备有很大的影响,由黄化苗或幼嫩的绿叶为材料分离细胞核,进行胶块包埋是制备大分子量核DNA的最佳条件。利用该法获得的DNA分子量在200kb－5．7Mb之间,主要集中在2．2～5．7Mb之间;每一胶块DNAE量为18～20μg。与包埋原生质体制备大分子量核DNA的方法相比,该方法获得的DNA纯度较高,去除了大部分细胞器DNA的污染;易于被限制性内切酶部分和完全消化,其消化结果具可重复性。该方法操作简单、适用植物种类广泛,用该方法从水稻（OryzasativaL．）、苹果（MaluspumilaMill．）、大豆（Glycinemax（L．）Merr．）、玉米（ZeamaysL．）等多种植物材料中成功地制备了大分子量核DNA。该方法制备的核DNA适用于植物的脉冲交变电泳基因组分析和构建人工细菌染色体文库和人工酵母染色体文库。     

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.