The second respiratory chain of Candida parapsilosis: a comprehensive study.

The yeast C. parapsilosis CBS7157 is strictly dependent on oxidative metabolism for growth since it lacks a fermentative pathway. It is nevertheless able to grow on high glucose concentrations and also on a glycerol medium supplemented with antimycin A or drugs acting at the level of mitochondrial protein synthesis. Besides its normal respiratory chain C. parapsilosis develops a second electron transfer chain antimycin A-insensitive which allows the oxidation of cytoplasmic NAD(P)H resulting from glycolytic and hexose monophosphate pathways functioning through a route different from the NADH-coenzyme Q oxidoreductase described in S. cerevisiae or from the alternative pathways described in numerous plants and microorganisms. The second respiratory chain of C. parapsilosis involves 2 dehydrogenases specific for NADH and NADPH respectively, which are amytal and mersalyl sensitive and located on the outer face of the inner membrane. Since this antimycin A-insensitive pathway is fully inhibited by myxothiazol, it was hypothesized that electrons are transferred to a quinone pool that is different from the classical coenzyme Q-cytochrome b cycle. Two inhibitory sites were evidenced with myxothiazol, one related to the classical pathway, the other to the second pathway and thus, the second quinone pool could bind to a Q-binding protein at a specific site. Elimination of this second pool leads to a fully antimycin A-sensitive NADH oxidation, whereas its reincorporation in mitochondria allows recovery of an antimycin A-insensitive, myxothiazol sensitive NADH oxidation. The third step in this second respiratory chain involves a specific pool of cytochrome c which can deliver electrons either to a third phosphorylation site or to an alternative oxidase, cytochrome 590. This cytochrome is inhibited by high cyanide concentrations and salicylhydroxamates.

Detection of Toxoplasma gondii in aqueous humour by the polymerase chain reaction.

Ocular toxoplasmosis is the most frequent infectious cause of chorioretinal inflammation in immunocompetent patients. Nowadays, the biological diagnosis of ocular toxoplasmosis requires serological tests and anterior chamber puncture to detect the local production of specific antibodies. A new technique is described to detect Toxoplasma in aqueous humour by a polymerase chain reaction in which the target is a specific ribosomal DNA segment. Sixty eight patients (71 eyes) were included; 59 (83%) eyes were suspected of having ocular toxoplasmosis. Of these 59 eyes, 15 (25.4%) had characteristic fundus lesions with obvious intraocular inflammation signs and 44 (75%) had retinal scar of ocular toxoplasmosis without clinically detectable inflammation. Twelve (17%) eyes had uveitis of non-Toxoplasma origin and constituted the control group. The parasite was present in aqueous humour in 20 (33.8%) cases. No false positives were detected. The sensitivity of the test is reduced by the low numbers of the sample. The combination of this technique with Witmer-Desmonts coefficient increases the probability of making a biological diagnosis of ocular toxoplasmosis. The physiopathological value of this technique is emphasised and the presence of tachyzoites in the anterior chamber is suggested. This should be a very promising technique for the diagnosis of ocular toxoplasmosis.

[Contribution of gene amplification in the biological diagnosis of toxoplasmosis]. / Apport de l'amplification génique au diagnostic biologique de la toxoplasmose.

Biological diagnosis of toxoplasmosis is generally based on indirect arguments (serology). In cases of immaturity or of immune depression, however, evidence of the parasite has to be obtained. This involves time-consuming or relatively insensitive culture techniques. Molecular biology, and more particularly the polymerase chain reaction gene amplification technique, makes it possible to identify an extremely small quantity of parasites in a complex biological fluid in a few hours. We summarized our experience with an original technique using toxoplasma ribosomal DNA as the target. Its use in the amniotic fluid provides a distinct improvement in antenatal diagnosis and is fast becoming the technique of reference. Difficult cases have been solved by its application in ophthalmology. Finally, in immunodepressed patients, especially in cases of acquired immunodeficiency syndrome, assessment is currently ongoing. The results obtained so far, especially in the analysis of the cerebral spinal fluid, are encouraging.

The alternative respiratory pathway of the yeast Candida parapsilosis: oxidation of exogenous NAD(P)H.

The yeast Candida parapsilosis possesses two routes of electron transfer from exogenous NAD(P)H to oxygen. Electrons are transferred either to the classical cytochrome pathway at the level of ubiquinone through an NAD(P)H dehydrogenase, or to an alternative pathway at the level of cytochrome c through another NAD(P)H dehydrogenase which is insensitive to antimycin A. Analyses of mitoplasts obtained by digitonin/osmotic shock treatment of mitochondria purified on a sucrose gradient indicated that the NADH and NADPH dehydrogenases serving the alternative route were located on the mitochondrial inner membrane. The dehydrogenases could be differentiated by their pH optima and their sensitivity to amytal, butanedione and mersalyl. No transhydrogenase activity occurred between the dehydrogenases, although NADH oxidation was inhibited by NADP+ and butanedione. Studies of the effect of NADP+ on NADH oxidation showed that the NADH:ubiquinone oxidoreductase had Michaelis-Menten kinetics and was inhibited by NADP+, whereas the alternative NADH dehydrogenase had allosteric properties (NADH is a negative effector and is displaced from its regulatory site by NAD+ or NADP+).

Detection of Toxoplasma gondii by PCR and tissue culture in cerebrospinal fluid and blood of human immunodeficiency virus-seropositive patients.

To investigate whether both tissue culture and PCR on a sequence from the repetitive rDNA could contribute to the diagnosis of toxoplasmosis, blood samples and, if they were available, cerebrospinal fluid (CSF) and aqueous humor samples from 72 human immunodeficiency virus-seropositive patients with suspected toxoplasmosis were prospectively tested. For 10 patients with fever of unknown origin but without confirmed toxoplasmosis, no Toxoplasma gondii was detected. For two patients with confirmed toxoplasmic uveitis, only PCR of aqueous humor samples was positive. Of 60 patients (48 with CSF samples) with neurological signs, 25 (from 13 of whom CSF samples were available) had confirmed cerebral toxoplasmosis and 10 had a positive PCR of CSF and/or blood samples, while for 1 patient culture of the CSF sample was also positive. Unlike tissue culture, PCR of rDNA is of value for the detection of cerebral toxoplasmosis in human immunodeficiency virus-seropositive patients, provided that both CSF and blood samples are available (sensitivity, 76.9%; specificity, 100%).