Solution structure of human saposin C in a detergent environment

Saposin C is a lysosomal, membrane-binding protein that acts as an activator for the hydrolysis of glucosylceramide by the enzyme glucocerebrosidase. We used high-resolution NMR to determine the three-dimensional solution structure of saposin C in the presence of the detergent sodium dodecyl sulfate (SDS). This structure provides the first representation of membrane bound saposin C at the atomic level. In the presence of SDS, the protein adopts an open conformation with an exposed hydrophobic pocket. In contrast, the previously reported NMR structure of saposin C in the absence of SDS is compact and contains a hydrophobic core that is not exposed to the solvent. NMR data indicate that the SDS molecules interact with the hydrophobic pocket. The structure of saposin C in the presence of SDS is very similar to a monomer in the saposin B homodimer structure. Their comparison reveals possible similarity in the type of protein/lipid interaction as well as structural components differentiating their quaternary structures and functional specificity.     

The O-chain structure from the LPS of the bacterium Naxibacter alkalitolerans YIM 31775T

The O-chain polysaccharide of the lipopolysaccharide from the bacterium Naxibacter alkalitolerans strain YIM 31775(T) was characterized. The structure was studied by means of chemical analysis and 2D NMR spectroscopy and shown to be built up by the following tetrasaccharide repeating unit: -->3)-alpha-D-FucpNAc-(1-->2)-beta-D-Quip3NHBu-(1-->2)-alpha-D-Rhap-(1-->)-beta-D-Galp-(1--> where HBu is hydroxy-butanoyl.     

Towards the Burden of Human Leptospirosis: Duration of Acute Illness and Occurrence of Post-Leptospirosis Symptoms of Patients in The Netherlands

Background

Leptospirosis is a global zoonotic disease. Although important for the assessment of the burden of leptospirosis, data on the duration of the illness and the occurrence of post-leptospirosis complaints are not well documented. Hence the main objective of this study was to estimate the occurrence of persistent complaints and duration of hospital stay in laboratory confirmed leptospirosis patients in the Netherlands during 1985 to 2010. Additionally, several risk factors potentially impacting on the occurrence of post-leptospirosis complaints were investigated.

Methods/Principal Findings

The duration of the acute phase of leptospirosis was 16 days (IQR 12–23); 10 days (IQR 7–16) were spent hospitalized. Eighteen fatal cases were excluded from this analysis. Complaints of leptospirosis patients by passive case investigations (CPC) derived from files on ambulant consultations occurring one month after hospital discharge, revealed persistent complaints in 108 of 236 (45.8%) laboratory confirmed cases. Data on persistent complaints after acute leptospirosis (PCAC), assessed in 225 laboratory confirmed leptospirosis cases collected through questionnaires during 1985-1993, indicated 68 (30.2%) PCAC cases. Frequently reported complaints included (extreme) fatigue, myalgia, malaise, headache, and a weak physical condition. These complaints prolonged in 21.1% of the cases beyond 24 months after onset of disease. There was no association between post-leptospirosis complaints and hospitalization. However, individuals admitted at the intensive care unit (ICU) were twice as likely to have continuing complaints after discharge adjusting for age and dialysis (OR 2.0 95% CI 0.8-4.8). No significant association could be found between prolongation of complaints and infecting serogroup, although subgroup analysis suggest that infection with serogroups Sejroe (OR 4.8, 95%CI 0.9-27.0) and icterohaemorrhagiae (OR 2.0, 95%CI 0.9-4.3 CI) are more likely to result in CPC than infections with serogroup Grippotyphosa.

Conclusion/Significance

In addition to the acute disease, persistent complaints have an impact on the burden of leptospirosis.     

Characteristics of in vivo murine erythropoietic response to sodium orthovanadate

Current knowledge about the effects of vanadium compounds on erythropoiesis is still reduced and even contradictory. The aim of this work was to evaluate the in vivo effects of a single dose of sodium orthovanadate (OV, 33 mg/kg i.p.) on CF-1 mice in a time course study (0-8 days). Murine erythropoiesis was assessed through a combinatory of experimental approaches. Classical peripheral and bone marrow (BM) hematological parameters were determined. Erythroid maturation in blood stream and hemopoietic tissues (59Fe uptake assays), BM erythroid progenitor frequency (clonogenic assays) and erythroid crucial protein expressions for commitment and survival: GATA-1, erythropoietin receptor (Epo-R) and Bcl-xL (immunoblottings) were evaluated. Neither BM cellularities nor BM viabilities changed noticeably during the study. Peripheral reticulocytes showed a biphasic increment on days 2 and 8 post-OV. hematocrits enhanced transiently between days 2 and 4. 59Fe uptake percentages enhanced in peripheral blood nearly two-fold over control values between 4 and 8 days (p<0.01) without changes in BM and spleen. Additionally, mature erythroid BM compartments: polychromatophilic erythroblasts and orthochromatic normoblasts increased by the eighth day. BFU-E colonies remained near basal values during the whole experience, whilst CFU-E colonies raised 60% over control at 8 days post-OV (p<0.05). GATA-1 and Epo-R were significantly over-expressed from the third until the end of the experimental protocol (p<0.01). Surprisingly, Bcl-xL showed a constitutive expression pattern without changes during the experience. Experimental data let us suggest that OV does not to cause bone marrow cytotoxicity and that it accelerates maturation of BM committed erythroid precursors. Moreover, there are significant correlations among erythroid-related protein expressions: GATA-1 and Epo-R and the frequency of CFU-E. In addition, Bcl-xL expression invariance during the time course study would indicate that the stimulatory effect of OV treatment on erythropoiesis was mainly exerted on the maturation of red cell precursors rather than on the antiapoptosis of erythroid terminal progenitors.     

The Gastroprotective Effect of Menthol: Involvement of Anti-Apoptotic,Antioxidant and Anti-Inflammatory Activities

The aim of this research was to investigate the anti-apoptotic, antioxidant and anti-inflammatory properties of menthol against ethanol-induced gastric ulcers in rats. Wistar rats were orally treated with vehicle, carbenoxolone (100 mg/kg) or menthol (50 mg/kg) and then treated with ethanol to induce gastric ulcers. After euthanasia, stomach samples were prepared for histological slides and biochemical analyses. Immunohistochemical analyses of the cytoprotective and anti-apoptotic heat-shock protein-70 (HSP-70) and the apoptotic Bax protein were performed. The neutrophils were manually counted. The activity of the myeloperoxidase (MPO) was measured. To determine the level of antioxidant functions, the levels of glutathione (GSH), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and superoxide dismutase (SOD) were measured using ELISA. The levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and the anti-inflammatory cytokine interleukin-10 (IL-10) were assessed using ELISA kits. The menthol treated group presented 92% gastroprotection compared to the vehicle-treated group. An increased immunolabeled area was observed for HSP-70, and a decreased immunolabeled area was observed for the Bax protein in the menthol treated group. Menthol treatment induced a decrease in the activity of MPO and SOD, and the protein levels of GSH, GSH-Px and GR were increased. There was also a decrease in the levels of TNF-α and IL-6 and an increase in the level of IL-10. In conclusion, oral treatment with menthol displayed a gastroprotective activity through anti-apoptotic, antixidant and anti-inflammatory mechanisms.     

Increased demand for NAD+ relative to ATP drives aerobic glycolysis

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Phospholipid base exchange enzyme activity in sarcolemmal membranes from the heart of cardiomyopathic hamsters

The activity of phospholipid base exchange enzymes has been evaluated in cardiac sarcolemmal membranes from Syrian Golden hamsters and from a hamster strain (UM-X7.1) characterized by a genetic form of hypertrophic cardiomyopathy. No choline base exchange activity and only a little serine base exchange activity were detected, whereas the ethanolamine base exchange enzyme was found highly active in membranes from both strains. For this reason, the present study is focussed on the ethanolamine base exchange enzyme. The apparent Km for ethanolamine of ethanolamine base exchange enzyme from Syrian Golden membranes and from UM-X7.1 strain membranes are 18 and 32 μM, respectively. The specific activity of the sarcolemmal ethanolamine base exchange enzyme is lower in the UM-X7.1 strain than in Syrian Golden hamsters. The calcium-dependence of the enzyme appears different when the membranes from the two strains are compared. Indeed, after removal of the membrane-bound divalent cations, comparable activities are found in both membrane preparations, whereas, upon addition of Ca²⁺ to the incubation mixtures, the activity of the enzyme is enhanced in the membranes from Syrian Golden strain more than in those from UM-X7.1 strain. The cholesterol content of sarcolemmal membranes is higher in the cardiomyopathic strain than in the Syrian Golden hamsters. A possible relation between changes of the membrane lipid composition and of the ethanolamine base exchange activity is discussed.     

Formation of S-(carboxymethyl)-cysteine in rat liver mitochondrial proteins: effects of caloric and methionine restriction

Maillard reaction contributes to the chemical modification and cross-linking of proteins. This process plays a significant role in the aging process and determination of animal longevity. Oxidative conditions promote the Maillard reaction. Mitochondria are the primary site of oxidants due to the reactive molecular species production. Mitochondrial proteome cysteine residues are targets of oxidative attack due to their specific chemistry and localization. Their chemical, non-enzymatic modification leads to dysfunctional proteins, which entail cellular senescence and organismal aging. Previous studies have consistently shown that caloric and methionine restrictions, nutritional interventions that increase longevity, decrease the rate of mitochondrial oxidant production and the physiological steady-state levels of markers of oxidative damage to macromolecules. In this scenario, we have detected S-(carboxymethyl)-cysteine (CMC) as a new irreversible chemical modification in mitochondrial proteins. CMC content in mitochondrial proteins significantly correlated with that of the lysine-derived analog N ^ε-(carboxymethyl)-lysine. The concentration of CMC is, however, one order of magnitude lower compared with CML likely due in part to the lower content of cysteine with respect to lysine of the mitochondrial proteome. CMC concentrations decreases in liver mitochondrial proteins of rats subjected to 8.5 and 25 % caloric restriction, as well as in 40 and 80 % methionine restriction. This is associated with a concomitant and significant increase in the protein content of sulfhydryl groups. Data presented here evidence that CMC, a marker of Cys-AGE formation, could be candidate as a biomarker of mitochondrial damage during aging.     

Cytogenetic and nucleolar meiotic cycle analyses in Dysdercus imitator Blöte, 1931 (Pyrrhocoridae, Heteroptera) from Argentina

So far, only seven and five species of Dysdercus from the Old and New Worlds, respectively, have been cytogenetically analysed. They all have holokinetic chromosomes and a pre-reductional type of meiosis. In the present study the chromosome complement, male meiosis and nucleolar meiotic cycle of Dysdercus imitator were analyzed. During male meiosis several cytogenetic features are remarkable, namely the presence of a long diffuse stage after pachytene, the finding of one or two ring bivalents per cell in almost all specimens, and the presence of several prenucleolar bodies lasting up to telophase II. The origin and function of these prenucleolar bodies could be related to a particular physiological cycle of the meiocytes.     

Development of 5-hydroxypyrazole derivatives as reversible inhibitors of lysine specific demethylase 1

A series of reversible inhibitors of lysine specific demethylase 1 (LSD1) with a 5-hydroxypyrazole scaffold have been developed from compound 7, which was identified from the patent literature. Surface plasmon resonance (SPR) and biochemical analysis showed it to be a reversible LSD1 inhibitor with an IC₅₀ value of 0.23 µM. Optimisation of this compound by rational design afforded compounds with K_d values of <10 nM. In human THP-1 cells, these compounds were found to upregulate the expression of the surrogate cellular biomarker CD86. Compound 11p was found to have moderate oral bioavailability in mice suggesting its potential for use as an in vivo tool compound.