Multilocus microsatellite typing revealed high genetic variability of Leishmania donovani strains isolated during and after a Kala-azar epidemic in Libo Kemkem district, northwest Ethiopia

In 2004, an outbreak of kala-azar (KA) occurred for the first time in Libo Kemkem district, in the highland area of northwest Ethiopia. In order to track the possible origins of the outbreak parasites, we have investigated 19 strains of Leishmania donovani that were collected during (n = 6) and after (n = 13) the outbreak by using 14 highly polymorphic microsatellite markers. Unique microsatellite profiles were obtained for all strains from Libo Kemkem. When compared to those of L. donovani strains from different Ethiopian, Kenyan and Sudanese foci, by genetic distance and Bayesian clustering model analyses, most strains from Libo Kemkem grouped with strains from: (i) Humera and Metema in the lowlands and Belessa in the highland of Ethiopia, and (ii) Sudan, at different hierarchal levels. The strains from Libo Kemkem district were assigned at least to three genetically distinct clusters (A, B1 and B2) of which only one, cluster B2, consisted exclusively of strains from Libo Kemkem. The fact that most of the outbreak strains were found to be related to strains from well-known KA foci in northwest Ethiopia and Sudan might suggest multiple introductions of L. donovani strains from these foci into Libo Kemkem district.     

Alteration of hydrogen bonding in the vicinity of histidine 48 disrupts millisecond motions in RNase A

The motion of amino acid residues on the millisecond (ms) time scale is involved in the tight regulation of catalytic function in numerous enzyme systems. Using a combination of mutational, enzymological, and relaxation-compensated (15)N Carr-Purcell-Meiboom-Gill (CPMG) methods, we have previously established the conformational significance of the distant His48 residue and the neighboring loop 1 in RNase A function. These studies suggested that RNase A relies on an intricate network of hydrogen bonding interactions involved in propagating functionally relevant, long-range ms motions to the catalytic site of the enzyme. To further investigate the dynamic importance of this H-bonding network, this study focuses on the individual replacement of Thr17 and Thr82 with alanine, effectively altering the key H-bonding interactions that connect loop 1 and His48 to the rest of the protein. (15)N CPMG dispersion studies, nuclear magnetic resonance (NMR) chemical shift analysis, and NMR line shape analysis of point mutants T17A and T82A demonstrate that the evolutionarily conserved single H-bond linking His48 to Thr82 is essential for propagating ms motions from His48 to the active site of RNase A on the time scale of catalytic turnover, whereas the T17A mutation increases the off rate and conformational exchange motions in loop 1. Accumulating evidence from our mutational studies indicates that residues experiencing conformational exchange in RNase A can be grouped into two separate clusters displaying distinct dynamical features, which appear to be independently affected by mutation. Overall, this study illuminates how tightly controlled and finely tuned ms motions are in RNase A, suggesting that designed modulation of protein motions may be possible.     

Draft genome sequence of Streptomyces acidiscabies 84-104, an emergent plant pathogen

A draft genome sequence of the plant pathogen Streptomyces acidiscabies 84-104, an emergent plant pathogen, is presented here. The genome is among the largest of streptomycetes, at more than 11 Mb, and encodes a 100-kb pathogenicity island (PAI) shared with other plant-pathogenic streptomycetes. The presence of this conserved PAI, and the remnants of a conserved integrase/recombinase at its 3' end, supports the hypothesis that S. acidiscabies emerged as a plant pathogen as a result of this acquisition.     

Taurine transporter gene expression in peripheral mononuclear blood cells of type 2 diabetic patients

Taurine acts as antioxidant, cell osmolyte, modulator of glucose metabolism, and plays a role in the retinal function. It is 10³-fold more concentrated in the intracellular than in the extracellular milieu due to a specific taurine-Na-dependent transporter (TauT), which is upregulated by hypertonicity, low extracellular taurine, or oxidative stress and acutely downregulated ‘in vitro’ by high glucose concentrations. Aim of this study was to investigate whether TauT expression was modified in mononuclear peripheral blood cells (MPC) of type 2 diabetic patients with or without micro/macrovascular complications. Plasma taurine, as well as other sulphur-containing aminoacids (assayed by HPLC) and TauT gene expression (assayed by real-time PCR analysis) were measured in MPC of 45 controls and of 81 age-and-sex matched type 2 diabetic patients with or without micro/macrovascular complications. Median value (interquartile range) of plasma taurine was significantly lower in diabetic patients than in controls [28.7 (13.7) μmol/l vs. 46.5 (20.3) μmol/l; P?<?0.05], while median TauT expression, in arbitrary units, was significantly higher in diabetics than in controls [3.8 (3.9) vs. 1 (1.3); P?<?0.05) and was related to HbA1c only in controls (r?=?0.34; P?<?0.05). Patients with retinopathy (n?=?25) had lower TauT expression than those who were unaffected [3.1 (2.8) vs. 4.1 (3.4); P?<?0.05], while persistent micro/macroalbuminuria was associated with unchanged TauT expression. A trend toward reduction in TauT expression was observed in patients with macroangiopathy [n?=?27; 3.3 (2.5) vs. 4 [3.7]; P?=?NS]. In conclusion, TauT gene is overexpressed in MPC of type 2 diabetic patients, while presence of retinopathy is specifically associated with a drop in TauT overexpression, suggesting its possible involvement in this microangiopathic lesion.     

A PTEN inhibitor displays preclinical activity against hepatocarcinoma cells

Phosphatase and tensin homolog (PTEN) gene is considered a tumor suppressor gene. However, PTEN mutations rarely occur in hepatocellular carcinoma (HCC), whereas heterozygosity of PTEN, resulting in reduced PTEN expression, has been observed in 32–44% of HCC patients. In the present study, we investigated the effects of the small molecule PTEN inhibitor VO-OHpic in HCC cells. VO-OHpic inhibited cell viability, cell proliferation and colony formation, and induced senescence-associated β-galactosidase activity in Hep3B (low PTEN expression) and to a lesser extent in PLC/PRF/5 (high PTEN expression) cells, but not in PTEN-negative SNU475 cells. VO-OHpic synergistically inhibited cell viability when combined with PI3K/mTOR and RAF/MEK/ERK pathway inhibitors, but only in Hep3B cells, and significantly inhibited tumor growth in nude mice bearing xenografts of Hep3B cells. Therefore, we demonstrated for the first time that VO-OHpic inhibited cell growth and induced senescence in HCC cells with low PTEN expression, and that the combination of VO-OHpic with PI3K/mTOR and RAF/MEK/ERK inhibitors resulted in a more effective tumor cell kill. Our findings, hence, provide proof-of-principle evidence that pharmacological inhibition of PTEN may represent a promising approach for HCC therapy in a subclass of patients with a low PTEN expression.     

Solution structure of an rRNA substrate bound to the pseudouridylation pocket of a box H/ACA snoRNA

Base pairing between the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs) and sequences in other eukaryotic RNAs target specific uridines for pseudouridylation. An RNA called HJ1 has been developed that interacts with the rRNA sequence targeted by the 5' pseudouridylation pocket of human U65 snoRNA the same way as intact U65 snoRNA. Sequences on both strands of the analog of the U65 snoRNP pseudouridylation pocket in HJ1 pair with its substrate sequence, and the resulting complex, called HJ3, is strongly stabilized by Mg(2+). The solution structure of HJ3 reveals an Omega-shaped RNA interaction motif that has not previously been described, which is likely to be common to all box H/ACA snoRNP-substrate complexes. The topology of the complex explains why the access of substrate sequences to snoRNPs is facile and how uridine selection may occur when these complexes form.     

Group IV Phospholipase A2α Controls the Formation of Inter-Cisternal Continuities Involved in Intra-Golgi Transport

The organization of intra-Golgi trafficking and the nature of the transport intermediates involved (e.g., vesicles, tubules, or tubular continuities) remain incompletely understood. It was recently shown that successive cisternae in the Golgi stack are interconnected by membrane tubules that form during the arrival of transport carriers from the endoplasmic reticulum. Here, we examine the mechanisms of generation and the function of these tubules. In principle, tubule formation might depend on several protein- and/or lipid-based mechanisms. Among the latter, we have studied the phospholipase A₂ (PLA₂)-mediated generation of wedge-shaped lysolipids, with the resulting local positive membrane curvature. We show that the arrival of cargo at the Golgi complex induces the recruitment of Group IVA Ca²⁺-dependent, cytosolic PLA₂ (cPLA₂α) onto the Golgi complex itself, and that this cPLA₂α is required for the formation of the traffic-dependent intercisternal tubules and for intra-Golgi transport. In contrast, silencing of cPLA₂α has no inhibitory effects on peri-Golgi vesicles. These findings identify cPLA₂α as the first component of the machinery that is responsible for the formation of intercisternal tubular continuities and support a role for these continuities in transport through the Golgi complex.     

Effects of HIV aspartyl-proteinase inhibitors on Leishmania sp.

In this work, we have found an antiproliferative effect on Leishmania sp. promastigotes and axenic amastigotes by the human immunodeficiency virus (HIV) aspartyl-proteinase inhibitors, Ac-Leu-Val-Phenylalaninal, Saquinavir mesylate and Nelfinavir, the latter two being used as part of antiretroviral therapy. This effect appears to be the result of cell division blockage. In addition, these drugs induced in culture a decrease in the percentage of co-infected HIV/Leishmania monocytes and amastigotes of Leishmania per macrophage. The finding of a dose-dependent inhibition of Leishmania promastigotes aspartyl-proteinase activity by these drugs allows us to propose this activity as the drug parasite target. A direct action of these HIV aspartyl-proteinase inhibitors on the parasite, would be correlated with the effect that highly active antiretroviral therapy have had in the decrease of HIV/Leishmania coinfection, opening an interesting perspective for new drugs research development based on this novel parasite proteinase family.     

Species-specific chemosignals evoke delayed excitation of the vomeronasal amygdala in freely-moving female rats

Male rat chemosignals attract females and influence their reproductive status. Through the accessory olfactory bulb and its projection target, the posteromedial cortical nucleus of the amygdala (PMCo), species-specific chemosignals detected by the vomeronasal organ (VNO) may reach the hypothalamus. To test this hypothesis in vivo, behavioural activation and neurotransmitter release in the PMCo were simultaneously monitored in freely moving female oestrus rats exposed to either rat or mouse urinary stimuli, or to odorants. Plasma levels of the luteinizing hormone were subsequently monitored. All stimuli induced an immediate behavioural activation, but only species-specific chemosignals led to a delayed behavioural activation. This biphasic behavioural activation was accompanied by a VNO-mediated release of the excitatory amino acids, aspartate and glutamate, in the PMCo. The late behavioural and neurochemical activation was followed by an increase in the levels of circulating luteinizing hormone. In conclusion, these data show that only species-specific chemosignals induce a delayed behavioural activation and excitatory activation of the PMCo, which is dependent on an intact VNO.