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.     

SSH和"越轨"RACE分子克隆技术

SSH和“越轨”RACE(step out RACE)均为创建干PCR抑制作用基础上的CDNA基因克隆新技术。SSH具有真阳性率高、不同丰度的mRNA都能被有效分离等优点,“越轨”RACE具有高灵敏度、低背景、操作简便等长处,这两项技术现已经成功应用干大量EST的分离、减数CDNA库的构建、差别表达新基因的分离,以及CDNA全长基因序列的克隆等方面。     

Studies on Trichomonads. I. The Metabolism of Tritrichomonas foetus and Trichomonads from the Nasal Cavity and Gecum of Swine

SUMMARY. The metabolism of Tritrichomonas foetus (strain, BP-1) and trichomonads from the nasal cavity and cecum of swine was studied manometrically under similar experimental conditions. At pH 6.4, quantitative and qualitative differences were observed. The cecal (probably T. suis ) and nasal trichomonad used glucose, galactose, fructose, mannose, lactose, sucrose, raffinose, and trehalose. T. foetus used all except lactose and raffinose. All three were inhibited by iodoacetate and arsenite. T. foetus and the nasal form were significantly inhibited by fluoride and 8-hydroxyquinoline, whereas the cecal trichomonad was not. At varied pH, all failed to oxidize Krebs' cycle intermediates. The amounts of oxygen consumed by T. foetus and the nasal trichomonad in the presence of lactate and pyruvate were at levels similar to those with disaccharides; the cecal trichomonad was indifferent toward both substances. Anaerobically, lactate and pyruvate increased the evolution of gas by all three trichomonads. Aerobic acid formation was demonstrated for all three forms. Anaerobically, metabolic CO₂ and gas(es) that were not absorbed by KOH were evolved by all three. Pure oxygen was inhibitory to glucose utilization and stimulatory to the endogenous respiration of all trichomonads; the nasal form was affected the least.
The writer believes that the cecal trichomonad is different from T. foetus and the nasal trichomonad of swine. The relationship between the nasal trichomonad and T. foetus remains in doubt.     

Comparison of Schaedler Agar and Trypticase Soy-Yeast Extract Agar for the Cultivation of Anaerobic Bacteria

Schaedler agar (SA) and Trypticase soy-yeast extract agar (TSYEA), both supplemented with rabbit blood (5%, v/v) and menadione (0.5 mg/liter), were compared with respect to quantitative recovery, quality of growth, and rapidity of growth of selected anaerobic bacteria. The media were stored for 2 to 4 days prior to use in an anaerobic glove box, where all subsequent bacteriological procedures were performed. After 24 hr of incubation, colonies of Clostridium cadaveris (C. capitovale), C. haemolyticum, C. novyi A, and C. perfringens were larger on SA than on TSYEA, and the appearance of C. novyi B colonies on SA at 24 hr antedated their appearance on TSYEA. Quantitative recovery of C. novyi B was improved on SA; recovery of the other clostridia tested was comparable on the two media (inconclusive results were obtained with C. novyi A). Rough colonial types of some of the clostridia emerged on SA. No appreciable differences in results with the two media were noted for Bacteroides fragilis, B. melaninogenicus, or Fusobacterium fusiforme.     

Life-History Characteristics and Colonizing Success in Plants

Successful plant colonizers are characterized by the attributesof weediness but exhibit a variety of life-history traits andgenetic systems. Colonizing success would depend on the abilityof exploiting niches in competition with earlier inhabitants.Within-population genetic variation plays a role in adaptationto new habitats. An experiment with hybrid derivatives froma cross between wild and cultivated soybean species proved thatthe ability to establish seedlings in semi-natural conditionswas genetically controlled, and was loosely correlated withvarious traits. A wild rice species, Oryza perennis, shows perennial-annualcontinuum among populations. The populations markedly differedin life-history traits and niche dimension, as conditioned byseasonal water regime and man's disturbance of the habitats.Intra-populational differentiation was also observed. The datasuggested that various life-history traits are selected as aset.     

Automated Device for Preparing Agar Media

An automated device which facilitates safe and effective agar media preparation without constant supervision is described.     

Multiple Syringe Inoculator for Agar Plates

The design and operation of a manually operated multiple syringe inoculator was described. Either 9 or 21 inoculations of constant volume could be made simultaneously. Up to 100 plates could be inoculated in 15 min with excellent reproducibility. No contact occurred between the inoculating needles and the agar surface. Construction was simple and inexpensive, with minimal maintenance.     

Comparison of Fecal Coliform Agar and Violet Red Bile Lactose Agar for Fecal Coliform Enumeration in Foods

A 24-h direct plating method for fecal coliform enumeration with a resuscitation step (preincubation for 2 h at 37 ± 1°C and transfer to 44 ± 1°C for 22 h) using fecal coliform agar (FCA) was compared with the 24-h standardized violet red bile lactose agar (VRBL) method. FCA and VRBL have equivalent specificities and sensitivities, except for lactose-positive non-fecal coliforms such as Hafnia alvei, which could form typical colonies on FCA and VRBL. Recovery of cold-stressed Escherichia coli in mashed potatoes on FCA was about 1 log unit lower than that with VRBL. When the FCA method was compared with standard VRBL for enumeration of fecal coliforms, based on counting carried out on 170 different food samples, results were not significantly different (P > 0.05). Based on 203 typical identified colonies selected as found on VRBL and FCA, the latter medium appears to allow the enumeration of more true fecal coliforms and has higher performance in certain ways (specificity, sensitivity, and negative and positive predictive values) than VRBL. Most colonies clearly identified on both media were E. coli and H. alvei, a non-fecal coliform. Therefore, the replacement of fecal coliform enumeration by E. coli enumeration to estimate food sanitary quality should be recommended.     

The fine structure of the axostyle and its associations with organelles in Trichomonads

The fine structure of the axostyle in the protists Tritrichomonas foetus and Monocercomonas sp is described using transmission electron microscopy after quick-freezing techniques and immunocytochemistry. The axostyle microtubules presents a lateral projection formed by two protofilaments in addition to the 13 protofilaments normally found in microtubules. The axostyle is associated with other cell structures such as hydrogenosomes, endoplasmic reticulum, sigmoid filaments and glycogen particles. The microtubules of the pelta-axostylar system are connected to each other by bridges regularly spaced with an interval of 9 nm. Labeling of the axostyle was observed after cell incubation with monoclonal antibodies recognizing alpha-tubulin and acetylated-tubulin.     

动物克隆核再程序化有关机理的研究进展

近年来,动物克隆技术发展迅速,不断有新的克隆动物面世。面对克隆效率、克隆动物存活率低的客观事实,人们对克隆有关机制做了很多探讨。在移植核的再程序化过程中,某些胞浆因子如核质原等在解除已分化细胞核染色质的抑制中发挥了关键的作用,同时移植核发生印记基因及非印记基因的去甲基化和重新甲基化的现象,这种染色质抑制作用的解除及基因的甲基化现象与移植核的去分化有着密切的联系,但其具体机制还不清楚。核移植技术中,供核与受体胞质的协调和核再程序化的关系得到大量研究,目前普遍认为为了使核再程序化进行得更完全,MⅡ期卵母细胞质是适宜的受体,而处于G0期或G1期的体细胞核是合适的核供体。     

Acetamide Agar Medium Selective for Pseudomonas aeruginosa

A synthetic base of acetamide and salts in agar permitted isolation of small numbers of Pseudomonas aeruginosa from swarming Proteus and other gram-negative species.     

Acetamide Agar for Differentiation of Nonfermentative Bacteria

An acetamide agar medium is described for use in the differentiation of nonfermentative gram-negative bacteria. With few exceptions, indicator reactions were rapid, intense, and clear-cut.     

Agar and agarose biotechnological applications

Agar, a phycocolloid obtained commercially from species of Gelidium and Gracilaria, has been known for several centuries; its earliest industrial application was in the preparation of solid microbiological media. The numerous techniques available for the purification of agar affect the characteristics of bacterial-grade agar. The availability of agarose, that fraction of agar with the lowest possible charge, has enhanced the utilzation of this phycocolloid. The process of gelation of agarose is discussed and the applications of agarose gels in different types of chromatography are summarized. Agarose has many and diverse important applications in biotechnology. These uses, and newly-developed ones, can be expected to increase the demands for high-quality agarose in the rapidly expanding field of biotechnology.     

Method for Staining and Preserving Agar Gel Diffusion Plates

An improved method for the preservation of agar gel precipitin lines has been developed.     

Isolation of Shigellae: VIII. Comparison of Xylose Lysine Deoxycholate Agar, Hektoen Enteric Agar, Salmonella-Shigella Agar, and Eosin Methylene Blue Agar with Stool Specimens

Two enrichment broths and four plating media were compared for efficiency of detection of enteric pathogens from 1,597 stool specimens. Of 170 salmonellae isolated from the composite of all methods, direct streaking yielded but 54%, whereas enrichment in gram-negative broth found 87% and Selenite-F broth 97%. By contrast, gram-negative broth produced 100% of the 17 shigellae, Selenite-F broth but 77%, and direct streaking only 59%. Thus, enrichment methods produced almost twice the number of both pathogens as direct streaking. Comparison of the plating media revealed xylose lysine deoxycholate agar (XLD) and Hektoen enteric agar to be equal in their abilities to find both pathogens. Both were moderately better than Salmonella-Shigella agar and markedly superior to eosin methylene blue agar. XLD fround 83% of salmonellae produced by the composite of four media and 90% of the shigellae. Hektoen enteric agar found 80% of both. Salmonella-Shigella agar detected 74 and 68%, respectively, and eosin methylene blue agar only 42 and 63%. The numbers of false positives accruing to each medium, however, showed Hektoen enteric and Salmonella-Shigella agars to produce more than twice as many false-positive plates as XLD. Similarly, Selenite-F broth resulted in many more false-positives for all plating media than did gram-negative broth. Consequently, the index of validity, which equates successful isolation of pathogens with total pickings, favored XLD and gram-negative broth as the media of choice, with direct streaking the poorest method by all counts.