Cloning, expression and functional characterisation of a peroxiredoxin from the potato cyst nematode Globodera rostochiensis

We report the cloning, expression and functional characterisation of a peroxidase belonging to the peroxiredoxin family from the potato cyst nematode Globodera rostochiensis, the first molecule of this type from any nematode parasitic on plants. The G. rostochiensis peroxiredoxin catalyses the breakdown of hydrogen peroxide, but not cumene or t-butyl hydroperoxide, in a trypanosomatid reducing system comprising trypanothione reductase, trypanothione and tryparedoxin. In common with its homologues from Onchocerca volvulus and Brugia malayi, the G. rostochiensis enzyme is present on the surface of invasive and post-infective juveniles despite the apparent lack of a cleavable N-terminal signal peptide. The possibility that the G. rostochiensis peroxiredoxin plays a role in protection of the parasite from plant defence responses is discussed.     

Retrograde Inversion Stripping as a Complication of the Clarivein? Mechanochemical Venous Ablation Procedure

The endovenous revolution has accelerated the development of new techniques and devices for the treatment of varicose veins. The ClariVein^® mechanochemical ablation device offers tumescentless treatment with a rotating ablation tip that can theoretically become stuck in tissue. We present the first report of retrograde stripping of the small saphenous vein without anaesthesia following attempted use of the ClariVein^® device, without adverse sequelae.     

"Subversive" substrates for the enzyme trypanothione disulfide reductase: alternative approach to chemotherapy of Chagas disease

The trypanosomatid flavoprotein disulfide reductase, trypanothione reductase, is shown to catalyze one-electron reduction of suitably substituted naphthoquinone and nitrofuran derivatives. A number of such compounds have been chemically synthesized, and a structure-activity relationship has been established; the enzyme is most active with compounds that contain basic functional groups in side-chain residues. The reduced products are readily reoxidized by molecular oxygen and thus undergo classical enzyme-catalyzed redox cycling. In addition to their ability to act as substrates for trypanothione reductase, the compounds are also shown to effectively inhibit enzymatic reduction of the enzyme's physiological substrate, trypanothione disulfide. Under aerobic conditions, trypanothione reductase is not inactivated by these redox-cycling substrates, whereas under anaerobic conditions the nitrofuran compounds cause irreversible inactivation of the enzyme. When tested for biological activity against Trypanosoma cruzi trypomastigotes, many of the test compounds were trypanocidal, and this activity correlated with their relative ability to act as substrates for trypanothione reductase. The activity of the enzyme with these redox-cycling derivatives constitutes a subversion of its normal antioxidant role within the cell. For this reason these compounds may be termed "subversive" substrates for trypanothione reductase.     

Admission inflammatory markers and isolation of a causative organism in patients with spontaneous spinal infection

Introduction

The purpose of this study was to investigate the significance of the inflammatory markers on admission in the isolation of a causative pathogen in patients with spinal infection. Spinal infection is treated frequently at spinal units and can encompass a broad range of clinical entities. Its diagnosis is often delayed because of the difficulty of identifying the responsible pathogen.

Methods

Patients with spinal infection treated in our institution over a period of eight years were identified and their notes studied retrospectively. Admission C-reactive protein (CRP), white cell count (WCC) as well as co-morbidities and mode of pathogen identification were recorded. Overall, 96 patients were included in the study.

Results

The CRP levels on admission were correlated significantly with the overall potential for isolation of a pathogen (p<0.0001) and positive biopsy cultures (p=0.0016). Admission WCC levels were associated significantly with the overall potential for isolation of a pathogen (p=0.0003) and positive biopsy cultures (p=0.0023). Both CRP and WCC levels were significantly negatively correlated with the duration of the preceding symptoms (p=0.0003 and p<0.0001 respectively). Delay in presentation was significantly negatively correlated with organism isolation (p=0.0001). Multivariate analyses identified the delay in presentation as the strongest independent variable for organism isolation (p=0.014) in cases of spontaneous spinal infection when compared with the admission CRP level (p=0.031) and WCC (p=0.056).

Conclusions

In spontaneous spinal infection, delay in presentation is the strongest independent variable for organism isolation. High inflammatory marker levels on admission are a useful prognostic marker for the overall potential of isolating a causative organism either by blood cultures or by biopsy in patients with negative blood cultures. Furthermore, the admission inflammatory marker levels allow for treating surgeons to counsel their patients of the likelihood of achieving a positive microbiological yield from biopsy.     

Assessing the Essentiality of Leishmania donovani Nitroreductase and Its Role in Nitro Drug Activation

The nitroimidazole fexinidazole has potential as a safe and effective oral drug therapy for the treatment of visceral leishmaniasis. To date, nitroheterocyclics have not been used in the treatment of leishmaniasis, and relatively little is known about their mechanism of action. In African trypanosomes, nitro drugs are reductively activated by a type I nitroreductase (NTR), absent in mammalian cells. Modulation of nitroreductase levels in Trypanosoma brucei directly affected sensitivity to nitro compounds, with reduced concentrations of the enzyme leading to moderate nitro drug resistance. In view of the progression of fexinidazole into clinical development for visceral leishmaniasis, here we assess the essentiality of the nitroreductase in Leishmania donovani and the effect of modulating nitroreductase levels on susceptibility to fexinidazole. The failure to directly replace both endogenous copies of the NTR gene, except in the presence of an ectopic copy of the gene, suggests that the NTR gene is essential for the growth and survival of L. donovani promastigotes. Loss of a single chromosomal copy of the L. donovani NTR gene resulted in parasites that were mildly resistant (<2-fold) to the predominant in vivo metabolite of fexinidazole, while parasites overexpressing NTR were 18-fold more susceptible. These data confirm that Leishmania NTR plays a pivotal role in fexinidazole activation. Reliance on a single enzyme for prodrug activation may leave fexinidazole vulnerable to the emergence of drug resistance. However, the essentiality of the NTR in L. donovani promastigotes, combined with the limited resistance shown by NTR single knockout cells, suggests that the potential for the spread of NTR-based resistance to fexinidazole may be limited.     

Pelvic ultrasound findings in different forms of sexual precocity

Recently produced reference curves for various ultrasound dimensions were used to retrospectively assess 67 pelvic ultrasound scans carried out at the initial presentation in girls with sexual precocity. At presentation the group with precocious puberty had significantly increased uterine lengths and ovarian volumes compared with the normal population, and a significantly increased fundal–cervical ratio. Ovarian volume was also significantly increased in thelarche and thelarche variant. The fundal–cervical ratio was significantly increased in thelarche variant. There was considerable overlap between individuals with sexual precocity and normal subjects. The ultrasound findings that best discriminated early or precocious puberty from other forms of sexual precocity were the presence of a midline endometrial echo, and a uterine length above the 97th centile for age. An entirely normal pelvic ultrasound at presentation did not rule out the possibility of precocious puberty.     

Uptake of the nitroimidazole drug megazol by African trypanosomes

Megazol, CL 64,855 (2-amino-5-[1-methyl-5-nitro-2-imidazolyl]-1,3, 4-thiazole) has been shown to be extremely effective in clearing experimental infections of African trypanosomes. An unusual amino-purine transporter termed P2, implicated in the transport of both the diamidine and melaminophenyl arsenical classes of drug in Trypanosoma brucei, recognised chemical groups on compounds which are also present on megazol. Megazol interacted with this carrier protein, as judged by its ability to inhibit P2 adenosine transport and to abrogate in vitro arsenical-induced lysis in a dose-dependent manner. However, parasites resistant to melaminophenyl arsenical and diamidine drugs due to lack of the P2 transporter showed no resistance to megazol. This is because passive diffusion represented the major route of entry. Initial rates of uptake were not saturable within the limit of megazol's solubility and did not conform to thermodynamic precepts compatible with carrier-mediated uptake. Adenosine and other P2 transporter substrates, even at high concentration, had little impact on megazol uptake. Uptake was biphasic, with a very rapid equilibration across the membrane followed by a slower accumulation over time. The equilibration phase represented a simple passive diffusion, with the subsequent uptake probably being due to metabolism of the drug.     

Chemotherapy versus supportive care in advanced non-small cell lung cancer: improved survival without detriment to quality of life

BACKGROUND: In 1995 a meta-analysis of randomised trials investigating the value of adding chemotherapy to primary treatment for non-small cell lung cancer (NSCLC) suggested a small survival benefit for cisplatin-based chemotherapy in each of the primary treatment settings. However, the meta-analysis included many small trials and trials with differing eligibility criteria and chemotherapy regimens. METHODS: The aim of the Big Lung Trial was to confirm the survival benefits seen in the meta-analysis and to assess quality of life and cost in the supportive care setting. A total of 725 patients were randomised to receive supportive care alone (n = 361) or supportive care plus cisplatin-based chemotherapy (n = 364). RESULTS: 65% of patients allocated chemotherapy (C) received all three cycles of treatment and a further 27% received one or two cycles. 74% of patients allocated no chemotherapy (NoC) received thoracic radiotherapy compared with 47% of the C group. Patients allocated C had a significantly better survival than those allocated NoC: HR 0.77 (95% CI 0.66 to 0.89, p = 0.0006), median survival 8.0 months for the C group v 5.7 months for the NoC group, a difference of 9 weeks. There were 19 (5%) treatment related deaths in the C group. There was no evidence that any subgroup benefited more or less from chemotherapy. No significant differences were observed between the two groups in terms of the pre-defined primary and secondary quality of life end points, although large negative effects of chemotherapy were ruled out. The regimens used proved to be cost effective, the extra cost of chemotherapy being offset by longer survival. CONCLUSIONS: The survival benefit seen in this trial was entirely consistent with the NSCLC meta-analysis and subsequent similarly designed large trials. The information on quality of life and cost should enable patients and their clinicians to make more informed treatment choices.     

Influence of caloric restriction on the development of atherosclerosis in nonhuman primates: progress to date

Caloric restriction (CR) has been observed to retard aging processes and extend the maximum life span in rodents. In an effort to evaluate the effect of this nutritional intervention on physiologic variables in higher species, several nonhuman primate trials are ongoing. In particular, a study evaluating the independent effect of CR on the extent of atherosclerosis was initiated in 1993 in 32 adult cynomolgus monkeys. Therefore, the trial was designed to achieve identical cholesterol intake after animals were randomized to a control group or a calorie-restricted group (30% reduction from baseline caloric intake). The animals were routinely evaluated for glycated proteins, plasma insulin and glucose levels, insulin sensitivity, and specific measures for abdominal fat distribution by CT scans over a 4-year interval. The results from 4 years of intervention demonstrate that CR improves cardiovascular risk factors (such as visceral fat accumulation) and improves insulin sensitivity. In contrast to other primate studies with normolipidemic animals, CR had no independent effects on plasma lipid levels and composition in the presence of equivalent amounts of dietary cholesterol intake. Preliminary analysis of atherosclerotic lesion extent in the abdominal aorta has failed to demonstrate differences between control animals and CR animals. Follow- up studies are being conducted to determine the effect of CR on atherosclerosis extent in coronary and carotid arteries.