Overexpression in Escherichia coli and purification of pteridine reductase (PTR1) from a clinical isolate of Leishmania donovani

Pteridine reductase 1 (PTR1) is part of a novel metabolic pathway in Leishmania associated with folate metabolism. Its main function is to salvage pterins but a second one is to reduce folates. The novelty and possible uniqueness of the pathway in which PTR1 is involved opens the possibility of developing specific inhibitors, which in combination with dihydrofolate reductase inhibitors could be highly effective against Leishmania. In order to increase our understanding of this putative important chemotherapeutic target, we present here the cloning, overexpression and purification of this enzyme from a clinical isolate of Leishmania donovani causing kala azar in India. This recombinant enzyme will set the basis for inhibition studies as well as for structure-function relationships.     

Enhanced endothelial nitric oxide-synthase activity in mice infected with Trypanosoma brucei

Infection of humans with Trypanosoma brucei causes sleeping sickness, which is invariably fatal if left untreated. The course of infection is characterised, among others, by multiple organ damage including cardiovascular dysfunctions such as hypotension and breakdown of the blood-brain barrier. The latter eventually leads to the parasite invasion into central nervous system and ultimately to the death of the patient. Nitric oxide (NO) synthesised from L-arginine via endothelial NO-synthase (eNOS) is involved in the control of vascular tone and permeability. The present study explores the effect of T. brucei infection on the endothelium-dependent in vitro vasomotor response of isolated mouse aortas. Aorta rings were suspended in organ chambers for isometric tension recording. The endothelium-dependent NO-mediated relaxation in response to acetylcholine (10(-9) to 10(-5) M) was markedly enhanced in the infected mice compared to controls (P<0.05), whereas the endothelium-independent vasodilation to an exogenous NO-donor, sodium nitroprusside, was comparable in both groups. Norepinephrine-stimulated contraction was also comparable in the absence or presence of the NO-synthase inhibitor N(omega)-Nitro-L-arginine methyl ester (L-NAME; 10(-4)M) in both groups. The enhanced endothelium-dependent relaxation in the infected mice correlated well with a 3.5-fold increase in eNOS protein level in these aortas as compared to those of control mice (P=0.05). Thus, T. brucei infection enhances eNOS protein expression in the endothelium, causing a pronounced vasodilation. Overproduction of NO in trypanosomiasis may be involved in the observed generalised hypotension and in an increased vascular permeability that facilitates T. brucei invasion into surrounding tissues and its penetration into the central nervous system in later phases of infection.     

The Day-Hospital of the University Hospital,Bobo Dioulasso: An Example of Optimized HIV Management in Southern Burkina Faso

ObjectivesTo evaluate the epidemiological evolution of patients with HIV (PtHIV), between 2002 and 2012, in a day-hospital that became an HIV reference centre for south-west Burkina Faso.ResultsA total of 7320 patients have been treated at the centre since 2002; the active file of patients increased from 147 in 2002 to 3684 patients in 2012. Mean age was stable at 38.4 years and the majority were female (71%). The delay to initiation of antiretroviral (ARV) treatment after HIV diagnosis decreased from 12.9 months in 2002 to 7.2 months in 2012. The percentage of PtHIV lost to follow-up, untreated for HIV and deaths all decreased after 2005. Voluntary anonymous screening and/or an evocative clinical picture were the main reasons for HIV diagnosis, usually at a late stage (41.1% at WHO stage 3). Virological success increased due to a decrease in time to initiation of ARV treatment and an increase in percentage of patients treated (90.5% in 2012, mainly with 1^st line drugs). However, there was also a slight increase in the rate of therapeutic failures and the percentage of patients who progressed to 2^nd or 3^rd line-ARVs.ConclusionOur day-hospital is a good example of the implementation of a specialist centre for the management of PtHIV in a resource-limited country (Burkina Faso).     

Induction of phytochelatins and antioxidant defence system in <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Vigna radiata</Emphasis> in response to chromium treatments

Growth performance, chromium (Cr) accumulation potential and induction of antioxidative defence system and phytochelatins (PCs) were studied in hydroponically grown Brassica juncea (Indian mustard) and Vigna radiata (mungbean) at various levels of Cr treatments (0, 50, 100, 200 μM Cr). B. juncea accumulated twofolds and threefolds higher Cr in root and shoot, respectively than in V. radiata. Compared to B. juncea, V. radiata was found to be particularly sensitive to Cr as observed by the severity and development of Cr toxicity symptoms and decreased growth. Induction of PC and enzymes of antioxidant defence system were monitored as plant’s primary and secondary metal detoxifying responses, respectively. There was induction of PC and enzymes of antioxidant defence system in both the plants. PCs were induced significantly in roots and shoot of both the plants at all the levels of Cr treatments. Significantly higher activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) were observed in shoot of B. juncea than V. radiata at all the levels of Cr treatments. Induction of PCs along with antioxidant defence system in response to Cr stress suggests the cumulative role of PCs and antioxidants in conferring tolerance against accumulated Cr in B. juncea, and thereby signifies the suitability of this plant as one of the potential remediators of Cr.     

A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing

Virus-induced gene silencing (VIGS) is a rapid and robust method for determining and studying the function of plant genes or expressed sequence tags (ESTs). However, only a few plant species are amenable to VIGS. There is a need for a systematic study to identify VIGS-efficient plant species and to determine the extent of homology required between the heterologous genes and their endogenous orthologs for silencing. Two approaches were used. First, the extent of phytoene desaturase (PDS) gene silencing was studied in various Solanaceous plant species using Nicotiana benthamiana NbPDS sequences. In the second approach, PDS sequences from a wide range of plant species were used to silence the PDS gene in N. benthamiana. The results showed that tobacco rattle virus (TRV)-mediated VIGS can be performed in a wide range of Solanaceous plant species and that heterologous gene sequences from far-related plant species can be used to silence their respective orthologs in the VIGS-efficient plant N. benthamiana. A correlation was not always found between gene silencing efficiency and percentage homology of the heterologous gene sequence with the endogenous gene sequence. It was concluded that a 21-nucleotide stretch of 100% identity between the heterologous and endogenous gene sequences is not absolutely required for gene silencing.